Antitoxin and vaccine platform based on nodavirus VLPs

ABSTRACT

Antitoxin and vaccine compositions based on nodavirus VLPs are provided. Anthrax antitoxin and vaccine compositions are provided. Methods of treating toxins with VLP-based antitoxins are provided. Methods of raising an immune response with immunogen decorated VLPs are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The subject patent application claims priority to and benefit of each ofthe following prior patent applications: U.S. Ser. No. 60/967,918 filedSep. 7, 2007, by Young et al., entitled “A NOVEL ANTITOXIN AND VACCINEPLATFORM BASED ON NODAVIRUS VLPS”; U.S. Ser. No. 60/928,261 filed May 7,2007, by Young et al., entitled “A NOVEL ANTITOXIN AND VACCINE PLATFORMBASED ON NODAVIRUS VLPS”; U.S. Ser. No. 60/902,485 filed Feb. 20, 2007,by Young et al., entitled “A NOVEL ANTITOXIN AND VACCINE PLATFORM BASEDON NODAVIRUS VLPS”; and U.S. Ser. No. 60/901,791 filed Feb. 16, 2007, byYoung et al., entitled “A NOVEL ANTITOXIN AND VACCINE PLATFORM BASED ONNODAVIRUS VLPS.” Each of these prior applications is incorporated hereinby reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported in part by grants P01AI056013 and R01GM066087from the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The invention is in the field of antitoxins and vaccines based onnodavirus VLPs. Anthrax antitoxins and vaccines are preferredembodiments of the invention.

BACKGROUND OF THE INVENTION

A variety of diseases are modulated by bacterial and other toxins.Antitoxins to treat many such diseases exist, often as antibodiesagainst the relevant toxin molecule. For example, antibodies againstdiphtheria are raised (classically, in horses) and administered as anantitoxin against diphtheria. However, even where such antitoxins areavailable, they may not provide optimal protection, and often haveundesired side effects. Moreover, for some serious illnesses such asBacillus anthracis infection, effective antitoxins are not available atall.

Anthrax is caused by the spore-forming, gram-positive bacterium Bacillusanthracis ¹. The disease is elicited when spores are inhaled, ingestedor transmitted through open wounds in the skin. Inhalational anthrax isthe deadliest form of the disease primarily because it is difficult todiagnose in a timely manner. Disease symptoms are initially nonspecificand systemic dissemination of anthrax toxin can occur prior toantibiotic treatment². The deliberate release of B. anthracis spores inthe U.S. in 2001 with the ensuing human fatalities and enormous cleanupcosts has underscored the need for better detection, treatment andprevention of anthrax.

The toxic effects of anthrax are predominantly due to an AB-type toxinmade up of a single, receptor-binding B subunit and two enzymatic Asubunits³. The A subunits are edema factor (EF, 89 kD), an adenylatecyclase that raises intracellular cAMP levels⁴, and lethal factor (LF,90 kD), a zinc protease that cleaves mitogen-activated protein kinasekinases^(5,6). The receptor binding B subunit is protective antigen(PA), which is initially synthesized as an 83 kD precursor. Uponreceptor binding, PA83 is cleaved by furin into a 63 kD product, whichforms heptamers that bind EF to form edema toxin (EdTx) and LF to formlethal toxin (LeTx)³. Two anthrax toxin receptors, widely distributed onhuman cells, have been identified: anthrax toxin receptor/tumorendothelial marker 8 (ANTXR1)⁷ and capillary morphogenesis gene 2(ANTXR2)⁸. Although both receptors bind PA through a 200 amino acidextracellular von Willebrand factor A (VWA) domain, the VWA domain ofANTXR2 has a 1000-fold higher binding affinity for PA than the VWAdomain of ANTXR1. In addition, ANTXR2 has been shown to mediateintoxication in vivo⁹⁻¹¹. Recently, the LDL receptor-related proteinLRP6 was shown to function as a co-receptor for anthrax toxininternalization¹².

The potential use of anthrax and other diseases as weapons ofbioterrorism has prompted increased efforts to develop better antitoxinsand vaccines. In the case of anthrax, protective immunity to B.anthracis infection is conferred by antibodies against PA, which is theprimary component of anthrax-vaccine adsorbed (AVA; Biothrax), the onlycurrently licensed anthrax vaccine in the US. Although AVA is safe andeffective, it is molecularly ill-defined, can cause adverse reactionsand is administered in a lengthy immunization schedule (6 doses over 18months)¹³. A second-generation vaccine based on recombinant PA adsorbedon aluminum hydroxide as adjuvant is currently in development.Preliminary data indicate that it is less potent than AVA and it islikely that several immunizations will be required to confer protectionin humans¹⁴. Thus, the development of a well-characterized vaccine thatinduces rapid immunity after a single injection remains an importantgoal.

A general and widely adaptable vaccine platform to raise an immuneresponse against anthrax and/or other serious diseases would be highlydesirable. The present invention provides such a platform for use aseither an antitoxin or a vaccine platform.

SUMMARY OF THE INVENTION

The present invention provides a general and widely adaptable antitoxinand vaccine platform. Recombinant nodavirus-derived virus like particles(VLPs) display toxin binding domains, and can be used as a multivalentantitoxin. In addition, these VLPs can be decorated with toxin,providing a multivalent immunogen that can be used to raise an immuneresponse against the displayed toxin moieties. In one particularlyuseful example, anthrax antitoxins and immunogens are provided for usein treating or preventing anthrax infection.

Accordingly, in a first aspect, the invention provides an antitoxincomprising a nodavirus-derived virus like particle (VLP) that displays aheterologous toxin binding domain.

In one particularly preferred embodiment, the antitoxin comprises ananthrax antitoxin and the heterologous toxin binding domain comprises apolypeptide sequence that binds to a component of an anthrax toxin. Forexample, the polypeptide sequence optionally binds to anthrax protectiveantigen (PA). In this example, the polypeptide sequence can include a PAbinding subsequence of an extracellular von Willebrand factor A (VWA)domain, e.g., an ANTRX2 protein VWA domain. In one specific example, theANTRX2 VWA domain includes residues 38-218 of the VWA domain.

The VLP can be derived from any of a variety of nodaviruses. In onepreferred embodiment, the VLP is derived from Flock House Virus (FHV).FHV and other nodaviruses comprise loop domains on the capsid/coatproteins of the virus, into which heterologous polypeptide sequences canbe inserted. For example, the ANTRX2 VWA domain can be inserted in placeof residues 265-267 of a FHV coat protein of the VLP. Up to about 180copies of the heterologous polypeptide (e.g., toxin binding domain) canbe displayed on an exterior surface of the VLP.

In a related aspect, the invention provides an immunogenic compositioncomprising a nodavirus-derived virus like particle (VLP) that comprisesa heterologous immunogen binding domain, which binding domain is boundto a heterologous immunogen. This “decorated” VLP can be used as apolyvalent immunogen and can act as a potent vaccine against theimmunogen. In one preferred but non-limiting example, the binding domainincludes a VWA domain of capillary morphogenesis protein 2 (CMG-2, alsoknown as ANTRX2) and the heterologous immunogen is derived from ananthrax toxin (e.g., part or all of anthrax PA).

The heterologous immunogen that is used to decorate the VLP can bederived from more than one immunogen or more than one immunogenicdomain. For example, the immunogen can include a first domain that isbound by the binding domain, and a second domain that is heterologous tothe first domain. The first and second domains can be recombinantlyfused and expressed as a fusion protein. For example, the binding domaincan be derived from a VWA domain of CMG-2, while the first domain of theheterologous immunogen is derived from a part of anthrax PA that isrecognized (bound) by the VWA domain of CMG-2. The second domain of theheterologous immunogen is optionally derived from another vaccine targetof interest, e.g., a toxin protein other than PA, such as a ricin toxinor a botulinum toxin. Furthermore, compositions of the invention caninclude multivalent vaccine compositions that include two or moredifferent second domains, whether displayed on a single VLP, or multipleVLPs in a single injection formulation.

Thus, the invention also includes an immunogenic composition thatincludes a viral nanoparticle derived from a nodavirus that alsoincludes a heterologous immunogen binding domain. In this aspect, theviral nanoparticle is decorated with at least one immunogen, theimmunogen including a first domain that is bound by the heterologousimmunogen binding domain, and a second domain that is heterologous tothe first domain. For example, the viral nanoparticle can be derivedfrom flock house virus and the heterologous binding domain can include aVWA domain of capillary morphogenesis protein 2. The first domain of theat least one immunogen can include a portion of a PA domain of ananthrax toxin that is bound by the VWA domain, while the second domaincan be derived from a toxin protein such as a ricin toxin or botulinumtoxin.

Optionally, the immunogenic composition can be multivalent. For example,the viral nanoparticle can be decorated with at least two differentimmunogens. Thus, in one illustrative example, the two differentimmunogens each have a first domain and a second domain, with the firstdomain being the same for each of the at least two immunogens. Thesecond domain is different for each of the immunogens, and the firstdomain is heterologous to the second domain.

Alternately or additionally, the composition can provide a multivalentimmunogen by providing different nanoparticles. For example, thecomposition can include a second viral nanoparticle that is differentfrom the first viral nanoparticle, e.g., where the second viral particleis derived from a nodavirus and includes a second heterologous immunogenbinding domain. The second viral nanoparticle is optionally decoratedwith at least one second immunogen that includes a first domain that isbound by the second heterologous immunogen binding domain. The seconddomain of the second immunogen is heterologous to the first domain. Inone example, the second heterologous immunogen binding domain and thefirst heterologous immunogen binding domain are the same, the firstdomain of the first immunogen and the first domain of the secondimmunogen are the same, and the second domain of the first immunogen andthe second domain of the second immunogen are different, therebyproviding a multivalent immunogenic composition. For example, the firstand second immunogen binding domains can be derived from a VWA domain ofcapillary morphogenesis protein 2, while the first domain of the firstand second immunogens are derived from anthrax PA. The second domain ofthe first and second immunogens are independently selected from, e.g., atoxin other than an anthrax toxin, a ricin toxin and a botulinum toxin.

In any of the embodiments herein, steric crowding can limit the numberof heterologous immunogens that decorate the VLP. For example, when theVLP is decorated with bound anthrax PA, the VLP typically includes 180binding domains coupled to up to a maximum of about 120 heterologousimmunogens.

Accordingly, the invention provides an immunogenic composition thatincludes a nodavirus-derived VLP such as an FHV having a heterologousimmunogen derived from an anthrax toxin such as anthrax PA.

In a related aspect, the invention provides a method of antitoxintherapy to treat a toxin in a patient. The method includes identifying apatient in need of antitoxin therapy; and, administering to the patientan antitoxin that comprises a nodavirus-derived virus like particle(VLP) that comprises a heterologous toxin binding domain. The bindingdomain binds the toxin in the patient, thereby acting as an antitoxin.

A variety of applications are provided herein. The patient can be ahuman patient or a veterinary patient. In one aspect, the patient isinfected with anthrax and the binding domain binds to a component of ananthrax toxin. All of the features noted above with respect to antitoxincompositions are applicable here as well, e.g., the VLP can be derivedfrom FHV or another nodavirus, the binding domain can include aheterologous VWA domain of a ANTRX2 protein, bound by a PA domain of theanthrax toxin, etc.

Administration can be, e.g., by i.v. or i.p. administration of the VLPto the patient, or any other administration method that brings theantitoxin into contact with toxin in the patient. The VLP typicallybinds the toxin in a dose-dependent manner and the method can includeselecting a dosage regimen for administration of the VLP that is capableof ameliorating the effect of the toxin in the patient.

In a similar aspect, a method of vaccinating a patient with an immunogendecorated nodavirus derived VLP is provided. The method includes bindingan immunogen to an immunogen binding site displayed on the VLP; and,administering the resulting immunogen decorated VLP to a patient,thereby vaccinating the patient against the immunogen.

As with the preceding methods, the patient can be a human or veterinarypatient. Administration to the patient can be done via injection, e.g.,s.c. or i.m. injection.

The features described for vaccine compositions are applicable to thisembodiment as well. For example, the immunogen can be derived from ananthrax PA, the VLP can be derived from Flock House Virus or anothernodavirus and the heterologous immunogen binding site can include a VWAdomain of an ANTXR2 protein, etc.

Thus, in one embodiment, a method of vaccinating a patient againstanthrax infection is provided. The method includes providing acomposition comprising a nodavirus-derived VLP that comprises or isdecorated with an anthrax-derived immunogen; and, administering thecomposition to a patient, thereby vaccinating the patient against theanthrax-derived immunogen.

General methods of vaccinating a patient against infection are also afeature of the invention. The methods include, e.g., providing acomposition comprising a nodavirus-derived VLP that comprises animmunogen binding site recombinantly expressed as part of the VLP,wherein the binding site is bound, in the composition, to an immunogen,where the immunogen comprises a first domain that is bound by thebinding domain and a second domain that is heterologous to the firstdomain. The composition is administered to a patient, therebyvaccinating the patient against the immunogen.

Any of the features noted herein with respect to the relevantcompositions of the invention are applicable to these method embodimentsas well. Thus, for example, the toxin binding moiety can bind to anthraxPA, where the first domain is derived from an anthrax PA, while thesecond domain is derived from, e.g., ricin toxin or botulinum A toxin.Further any of the features noted in respect to multivalent immunogeniccompositions are applicable to this method as well, e.g., where the VLPcomprises a plurality of toxin binding moieties, and the compositioncomprises a plurality of different immunogens bound to the plurality oftoxin binding moieties. Multiple VLPs can also be administered toprovide multivalent protection, e.g., where the VLPs comprise differentimmunogens. Medicaments formulated for treatment by the methods hereinare a feature of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 Panels A-D. (a) Ribbon diagram of a single FHV coat proteinsubunit showing the surface-exposed loops into which the VWA domain ofANTXR2 was inserted. (b) Schematic diagram showing structure ofFHV-VWA_(ANTXR2) chimeric proteins 206 (top) and 264 (bottom). (c)Electrophoretic analysis of purified VLPs on a 10% Bis-Tris gel stainedwith Simply Blue (Invitrogen). (d) Electron micrographs ofgradient-purified wt and chimeric VLPs negatively stained with uranylacetate.

FIG. 2 provides a dose-response curve showing cell viability as afunction of antitoxin concentration.

FIG. 3 Panels A-D. Top panels A-B: Pseudoatomic models ofFHV-VWA_(ANTXR2) chimeras. Panels show surface views. Bottom panels C-D:In silico model of PA83 bound to the surface of FHV-VWA_(ANTXR2)chimeras.

FIG. 4A-F provide histograms showing results of antibody and lethaltoxin challenge responses for immunized rats.

FIG. 5 Panels A-C schematically show 3D surface-shaded reconstructionsof (a) FHV-VWA_(ANTXR2) chimera 206, (b) FHV-VWA_(ANTXR2) chimera 264and (c) wt FHV.

FIG. 6 Panels A-B. Pseudoatomic models of FHV-VWA_(ANTXR2) chimeras.

FIG. 7 Panels A-B. In silico model of PA83 bound to the surface ofFHV-VWA_(ANTXR2) chimeras.

FIG. 8 Panels A-B provide a gel photograph (panel A) and a quantitativehistogram showing the number of PA83 per chimera (Panel B).

FIG. 9 shows a schematic drawing of a way to modify a VNI (VLP) platformto present multiple immunogens to the immune system. As shown, thestrategy fuses PA with antigenic portions of, e.g., ricin or botulinumtoxins (e.g. PA-R and PA-B, respectively). These fusion proteins can bemultivalently arrayed on the surface of VNIs to generate combinationvaccines.

FIG. 10 schematically depicts a computational model of VNI-PA, VNI-PA-R,and VNI-PA-B. Domain 1 of PA (light purple in VNI-PA) is replaced withricin (red) and botulinum neurotoxin A (blue) protein domains,respectively, to form VNI-PA-R or VNI-PA-B. Construction of the PAfusion proteins is described in Example 2.

DETAILED DISCUSSION OF THE INVENTION

The invention provides nodavirus VLPs that comprise antitoxin activity,and that can also be decorated with an immunogen of interest to boost animmune response to the immunogen, e.g., in vaccine applications. TheVLPs provide a robust platform for the presentation of essentially anyimmunogen of interest, by including an immunogen binding domain in theVLP and decorating the resulting recombinant VLP with a correspondingimmunogen that is bound by the binding domain. The correspondingimmunogen can be the immunogen of interest, or the correspondingimmunogen can itself be a recombinant fusion of two or more moieties,e.g., an anchoring domain that is bound by the recombinant VLP and anadditional domain that includes an immunogen target of interest.Multivalent immunogenic compositions are also contemplated, in which oneor more VLP(s) in a vaccine composition is/are decorated with aplurality of different immunogens. The immunogens can include differentdomains of interest, e.g., in conjunction with a common anchoring domainthat is bound by the recombinant VLPs. These domains of interest caninclude, e.g., toxin proteins such as anthrax toxin, ricin toxin,botulinum toxin, or essentially any other immunogen of interest.

In one specific example, a VLP of the T=3 insect nodavirus Flock Housevirus (FHV) was produced that displays 180 copies of the von WillebrandA (VWA)-domain of capillary morphogenesis protein-2 (CMG-2) (also knownas ANTRX2 protein) on the surface of the VLP. ANTRX2 is a receptor foranthrax toxin and the VWA-domain represents the binding site forprotective antigen (PA). In this illustrative example, we replaced aminoacids 265-267 of the FHV coat protein with 181 amino acids representingresidues 38-218 of ANTRX2. These sites (in FHV or other similarnodaviruses) can be used for the insertion of other antitoxin/immunogenbinding domains of interest.

The modified coat protein was expressed in Spodoptera frugiperda cellsand Trichoplusia ni cells using a recombinant baculovirus vector. Theprotein spontaneously assembled into VLPs that packaged random RNA.Electron cryomicroscopy and three dimensional image reconstruction ofpurified VLPs confirmed that the VWA domains are displayed on thesurface of the VLPs. Computational modeling suggested that approximately120 monomers of anthrax PA can bind to each particle. Steric crowding inthis particular application prevents occupation of all 180 VWA domains.

Purified VWA VLPs functioned as a potent antitoxin in cell culture andprotected rats from lethal anthrax toxin challenge. In addition, VLPsdecorated with PA induced a robust antibody response against PA in rats,even in the absence of adjuvant. The response was significantly strongerthan that induced against monomeric PA in control rats. Rats vaccinatedwith VLP-PA complexes were completely protected from intravenouschallenge with anthrax lethal toxin whereas control rats that werevaccinated with monomeric PA did not survive.

FHV (or other nodavirus) VLPs displaying the VWA-domain of ANTRX2 showanthrax antitoxin activity in vivo and may therefore be useful as aneffective therapeutic for the treatment of infected individuals that donot respond to antibiotic treatment (or as an adjunct to antibiotictreatment).

Further, in the case of anthrax, there are no antitoxins previouslyavailable for use in treatment. The ease of synthesis and purificationof VLPs as described herein, the in vitro and in vivo stability and themultivalent display of the VWA domain represent advantages even relativeto the potential use of soluble ANTRX2 as an antitoxin.

Additionally, FHV-VWA VLPs decorated with PA serve as an excellentimmunogen to raise a potent antibody response to PA. This response issuperior to monomeric rPA that is currently in commercial development asan anthrax vaccine. The use of VLPs decorated with PA reduces the numberof immunizations required to achieve protection, which is a concern withthe current rPA vaccine. Thus, such complexes serve as a novel andhighly effective anthrax vaccine. Similar uses of VLPs to display otherimmunogens of interest should also result in a potent immune response;thus, the present invention, as well as providing an effective anthraxvaccine, also provides a general platform for vaccine development andproduction. As noted, for convenience, the immunogens of interest thatare used to decorate the VLP can include a common anchoring domain thatis bound by the VLP, in conjunction with essentially any immunogen ofinterest.

FHV-VWA VLPs decorated with PA can be used as a vaccine against anthrax.The polyvalent display of PA in the context of the virus particle leadsto induction of a very strong antibody response, even in the absence ofadjuvant, when compared to monomeric PA. In addition, the immunogen ismolecularly well-defined and characterized, in contrast to the anthraxvaccine that is currently in use. Another advantage pertains to the factthat subjects are protected from lethal toxin challenge after only twoinjections with immunogen. This is a significant improvement compared tothe lengthy immunization schedule with the currently available vaccine.Finally, using PA complexed to ANTRX2 as an antigen can lead toinduction of antibodies that crosslink PA to ANTRX2. Such antibodies maybe particularly useful in conferring protection against anthraxinfection. The PA-ANTRX2 interaction also inhibits PA from binding tocells in vivo, thus minimizing the possibility that, during postexposureimmunization, a PA immunogen could participate in intoxication.

Similar effects for VLPs that are decorated with one or more additionalimmunogens are expected; accordingly, the invention provides a universalplatform for vaccine development and production.

Making VLP Antitoxins

One aspect of the invention relates to the use of nodavirus-derived VLPsthat include heterologous molecular decoy moieties that bind to toxins.The resulting VLPs are antitoxins against the toxin, e.g., in a human orveterinary patient, reducing a toxic effect of the toxin. In onepreferred embodiment, coat protein of the nodavirus Flock House Virus(FHV) is used for the multivalent display of the Von Willebrand A (VWA)domain of capillary morphogenesis protein 2 (CMG-2; also known asANTRX2), a primary receptor for anthrax toxin. Sites for the insertionof the VWA domain in the coat protein were selected based on analysis ofthe high-resolution crystal structure of FHV. The resulting chimericvirus-like-particles (VLPs) can be generated in a baculovirus system andhave been shown to protect both cultured cells and live animals fromtoxic effects of anthrax protective antigen (PA) and lethal factor(anthrax toxin), indicating that the chimeric VWA domain binds PA,acting as a molecular decoy for anthrax toxin.

Nodavirus Derived VLPs

Nodaviruses such as FHV are non-enveloped, icosahedral (T=3) insectviruses of the family nodaviridae (Johnson et al. (1994) “Comparativestudies of T=3 and T=4 icosahedral RNA insect viruses,” Arch VirolSuppl. 9:497-512). For an introduction to nodaviruses, see Schneemann etal. (1998) “The structure and function of nodavirus particles: aparadigm for understanding chemical biology.” in Advances in virusresearch, Vol. 50 (eds. Maramorsch, K., Murphy, F. A. & Shatkin, A. J.)381-446 (Academic Press, San Diego). Nodaviruses have been described ininsects and fish, and are the etiological agent of significant diseasessuch as Viral Nervous Necrosis (VNN), also known as fish encephalitis(Samuelsen et al. (2006) “Viral and bacterial diseases of Atlantic codGadus morhua, their prophylaxis and treatment: a review,” Dis AquatOrgan. 71 (3):239-54). The Nodaviridae have bipartite RNA genomescomprising two separate single-stranded RNA molecules, designated RNA1and RNA2. In the natural viral life cycle, RNA1 and RNA2 are bothpackaged within the same virion. RNA1 encodes an RNA replicase, and RNA2encodes a capsid protein. In one representative example, Flock HouseVirus (FHV), RNA1 is 3.1 kb long and RNA2 is 1.4 kb.

Accordingly, flock house virus (FHV) is a bipartite, positive-strand RNAinsect nodavirus that encapsidates its two genomic RNAs (RNA1 and RNA2)in a single virion in a manner similar to other nodaviruses, such asBlack Beetle virus (BBV), Boolarra virus (BoV), Gypsy moth virus (GMV),and Manawatu virus (MwV). FHV provides one preferred nodavirus formodification according to the invention, although one of skill is ableto use other nodaviruses in a similar manner. Nodamura virus is anotheruseful example (see also, Am. J. Epidemiol. 86 (2), 271-285) as isPariacoto virus (see also, J. Virol. 74 (11):5123-5132).

The FHV capsid is composed of 180 subunits of a single type of coatprotein, and the icosahedral, solid shell capsid encapsidates abipartite, single-stranded RNA genome. The crystal structure of FHVparticles shows that the coat protein contains several surface-exposedloops that can be targeted for insertion of foreign proteins andpeptides. See Fisher & Johnson “Ordered duplex RNA controls capsidarchitecture in an icosahedral animal virus.” Nature 361, 176-179;Thiery et al. NODAVIRUS-VLP IMMUNIZATION COMPOSITION WO 2005/112994 A1;Hall RECOMBINANT NODAVIRUS COMPOSITIONS AND METHODS U.S. Pat. No.6,171,591; Ahlquist et al. (1994) “Protein-protein interactions andglycerophospholipids in bromovirus and nodavirus RNA replication” ArchVirol Suppl. 9:135-45. In general, the expression of nodavirus capsidproteins in cell culture, e.g., bacterial or insect cells, can producemature virus like particles (VLPs), e.g., using a recombinant expressionsystem that expresses the open reading frame of the RNA2 gene (e.g., ina baculovirus expression system). See, e.g., Oliveira et al. (2000)“Virus Maturation Targets the Protein Capsid to Concerted Disassemblyand Unfolding,” J. Biol. Chem. 275 (21):16037-16043. Several examples ofexpression systems that make VLPs using expression systems that expressNodavirus capsid proteins are available and can be adapted to thepresent invention. An example of a betanodavirus expression system otherthan FHV that generates VLPs is available for the Dragon grouper,Epinephelus lanceolatus, nervous necrosis virus (DGNNV); see, e.g., Luand Lin (2003) “Involvement of the terminus of grouper betanodaviruscapsid protein in virus-like particle assembly” Archives of Virology 148(2): 0304-8608.

Making Recombinant VLPs

The coat protein of a nodavirus such as FHV can be recombinantlyengineered to include essentially any polypeptide of interest. Thecrystal structure of FHV has been determined, and other nodaviruses canbe deduced by comparison to solved FHV structures or generalcrystallographic or other available virus structure determinationmethods. Nodaviruses such as FHV have surface loop domains into which apolypeptide of interest can be inserted for VLP display. As discussed indetail in the examples herein, preferred sites for insertion include twosurface-exposed loops at approximately amino acid positions thatcorrespond to 206 and 264 of FHV. In the examples herein, these sitesare shown to accommodate a 181 amino acid ANTXR2 VWA domain withoutdisrupting coat protein assembly into virus-like particles (VLPs) (FIG.1 a). In FHV-VWA_(ANTXR2) chimera 206, the VWA domain and a C-terminaltwo amino acid linker (Ala-Glu) replaced FHV coat protein residues207-208 (FIG. 1 b). In FHV-VWA_(ANTXR2) chimera 264, the VWA domainreplaced FHV residues 265-267. General regions that can be manipulatedfor insertion include Loop L1 (corresponding to amino acids 195-219(including, e.g., 201-213) of FHV coat protein), Loop L2 (correspondingto amino acids 263-277 of FHV coat protein), Loop L3 (corresponding toamino acids including e.g., 124-142 of FHV coat protein), Loop I1(corresponding to amino acids 107-110 of FHV coat protein), Loop 12(corresponding to amino acids 152-165 of FHV coat protein), and Loop 13(corresponding to amino acids 304-310 of FHV coat protein).

Insertions or deletions of heterologous proteins of interest at thesesites can be engineered into cDNAs corresponding to the RNA2 gene of therelevant nodavirus, e.g., in a baculovirus expression system forexpression in insect cells such as Trichoplusia ni cells or SpodopteraFrugiperda (e.g., IPLB-Sf21) cells.

cDNAs corresponding to an RNA2 nodavirus gene are engineered by standardcloning, mutagenesis or PCR based methods. Details regarding suchprocedures are found in standard references, e.g., general texts thatdescribe molecular biological techniques for the cloning andmanipulation of nucleic acids and production of encoded polypeptides.These include Sambrook et al., Molecular Cloning—A Laboratory Manual(3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 2001 (“Sambrook”) and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through the current date) (“Ausubel”) and Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger)). Thesetexts describe mutagenesis, the use of expression vectors, promoters andmany other relevant topics related to, e.g., the generation of clonesthat comprise nucleic acids of interest.

The present invention also relates to the production of transgenicorganisms, which may be, e.g., bacteria, yeast, fungi, animal or plantcells (or even whole organisms), transduced with the nucleic acids ofthe invention (e.g., nucleic acids encoding nodavirus capsid proteinsthat include heterologous sequences, as noted herein). A thoroughdiscussion of techniques relevant to bacteria, unicellular eukaryotesand cell culture generally is found in references enumerated herein andare briefly outlined as follows. Several well-known methods ofintroducing target nucleic acids into bacterial cells are available, anyof which may be used in the present invention. These include: fusion ofthe recipient cells with bacterial protoplasts containing the DNA,treatment of the cells with liposomes containing the DNA,electroporation, projectile bombardment (biolistics), carbon fiberdelivery, and infection with viral vectors (discussed further, below),etc. Bacterial or, e.g., insect cells can be used to amplify the numberof plasmids containing DNA constructs of this invention. The cells aregrown to log phase and the plasmids within the cells can be isolated bya variety of methods known in the art (see, for instance, Sambrook). Inaddition, a plethora of kits are commercially available for thepurification of plasmids from bacteria. For their proper use, follow themanufacturer's instructions (see, for example, EasyPrep™, FlexiPrep™,both from Pharmacia Biotech; StrataClean™, from Stratagene; and,QIAprep™ from Qiagen). The isolated and purified plasmids are thenfurther manipulated to produce other plasmids, used to transfect othercells or incorporated into organism specific vectors to infect cells ofinterest. Typical vectors contain transcription and translationterminators, transcription and translation initiation sequences, andpromoters useful for regulation of the expression of the particulartarget nucleic acid. The vectors optionally comprise generic expressioncassettes containing at least one independent terminator sequence,sequences permitting replication of the cassette in eukaryotes, orprokaryotes, or both, (e.g., shuttle vectors) and selection markers forboth prokaryotic and eukaryotic systems. Vectors are typically suitablefor replication and integration in prokaryotes, eukaryotes, or both.See, Giliman & Smith (1979) Gene 8:81; Roberts et al. (1987) Nature328:731; Schneider et al. (1995) Protein Expr. Purif. 6435:10; Ausubel,Sambrook, Berger (all infra). A catalogue of Bacteria and Bacteriophagesuseful for cloning is provided, e.g., by the ATCC, e.g., The ATCCCatalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds)published by the ATCC. Additional basic procedures for sequencing,cloning and other aspects of molecular biology and underlyingtheoretical considerations are also found in Watson et al. (1992)Recombinant DNA, Second Edition, Scientific American Books, NY. Inaddition, essentially any nucleic acid can be custom or standard orderedfrom any of a variety of commercial sources, such as the MidlandCertified Reagent Company (Midland, Tex.), The Great American GeneCompany (Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), OperonTechnologies Inc. (Alameda, Calif.) and many others.

Cell culture media appropriate for growing cells that comprise VLPs ingeneral are set forth in the previous references and, additionally, inAtlas and Parks (eds) The Handbook of Microbiological Media (1993) CRCPress, Boca Raton, Fla. Additional information for cell culture is foundin available commercial literature such as the Life Science ResearchCell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.)(“Sigma-LSRCCC”) and, e.g., the Plant Culture Catalogue and supplement(e.g., 1997 or later) also from Sigma-Aldrich, Inc (St Louis, Mo.)(“Sigma-PCCS”). The culture of animal cells is described, e.g., byFreshney (2000) Culture of Animal Cells: A Manual Of Basic TechniquesJohn Wiley and Sons, NY. Preferred cell culture media for insect cellculture (particularly applicable to baculovirus expression, discussed inmore detail below) is available from Expression Systems, Inc. (Davis,Calif.).

In addition to other references noted herein, a variety ofpurification/protein purification methods are well known in the art andcan be applied to VLP purification, including, e.g., those set forth inR. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982);Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc. N.Y. (1990); Sandana (1997)Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996)Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) The ProteinProtocols Handbook Humana Press, NJ; Harris and Angal (1990) ProteinPurification Applications: A Practical Approach IRL Press at Oxford,Oxford, England; Harris and Angal Protein Purification Methods: APractical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3rd Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and thereferences cited therein. Preferred methods of VLP purification includesucrose density purification, e.g., as described in the examples herein.

Further Details Regarding Baculovirus and Other Insect Cell ExpressionSystems

In one convenient aspect, recombinant nodavirus capsid protein can beexpressed to produce VLPs in cells using a baculovirus expressionsystem. The Baculoviridae infect many species of insects. Bacculoviridaehave strong promoters and a well-characterized genome that make themsuitable as expression vectors in insect cells. For example, the mostcommon invertebrate expression vector system is based on the AutographaCalifornica nuclear polyhedrosis virus (AcNPV), an insect baculovirusisolated from the Alfalfa looper. This virus replicates in the nucleusof many different lepidopteran insect cell lines. The baculovirusexpression vector system is commonly used to express genes derived fromother viruses (e.g., as in the present invention), as well as fromfungi, bacteria, plants, and animals. In this system, foreign genesplaced under the control of the strong polyhedrin promoter of the AcNPVare expressed at high levels in cultured lepidopteran (and many other)insect cells. In addition, Baculovirus have shown the ability to be usedas vectors for a variety of mammalian cell lines. The most widely usedlepidopteran cells for Baculovirus expression systems are the Sf9 andSf21 cell lines isolated from ovarian tissue of the fall army worm,Spodoptera frugiperda, and the High Five cell line, designatedBTITn-5B1-4, originally established from Trichoplusia ni embryonictissue. A variety of additional details regarding Baculovirus expressionsystems can be found on the internet at bacculovirus(dot)com and atexpressionsystems(dot)com, and in Baculovirus Expression Protocols(Methods in Molecular Biology, Vol 39) Christopher D. Richardson(Editor); (1998) Humana Pr; ISBN: 0896032728; O'Reilly et al.,Baculovirus Expression Vectors A Laboratory Manual, 1994 OxfordUniversity Press, and The Baculovirus Expression System: A LaboratoryGuide by Linda A. King, R. D. Possee; (1992) Chapman & Hall; ISBN:0412371502; and the references cited therein.

As applied to the present invention, FHV-like particles can be generatedby expressing FHV coat protein/heterologous domain chimeric genes in abaculovirus expression system. For a description of baculovirusexpression of nodavirus proteins, See, for example, Schneemann et al.(1993) “Use of recombinant baculoviruses in synthesis of morphologicallydistinct virus-like particles of flock house virus, a nodavirus.” J.Virol. 67, 2756-2763; O'Reilly et al. (1992) Baculovirus ExpressionVectors: A Laboratory Manual. W. H. Freeman and Co. New York; Vlak andKeus (1990) In Viral Vaccines. Wiley-Liss, Inc., New York. pp. 91-128;and Gallagher & Rueckert (1988) “Assembly-dependent maturation cleavagein provirions of a small icosahedral insect ribovirus.” J. Virol. 62,3399-3406. This allows for the large-scale production of chimericparticles that form into VLPs. For example, Trichoplusia ni orSpodoptera Frugiperda (e.g., IPLB-Sf21, or SF9) cells can be propagatedand infected, e.g., as described by Dong et al. (1998) “Particlepolymorphism caused by deletion of a peptide molecular switch in aquasi-equivalent virus” J. Virol. 72, 6024-6033 (1998). Other suitablecell lines include Manduca cell lines (e.g., Manduca Sexta cell lines),Estigmene cell lines (e.g., Estigmene acrea cell lines), Monarchbutterfly cell lines, Mamestra cell lines (e.g., Mamestra brassicae celllines), Dipteran cell lines, Drosophila insect cell line Schneider 2(S2), and cell line Kc1.

Accordingly, a variety of proteins have been produced by standardbaculovirus expression vector system, and VLPs of the invention can bemade by such protocols. Technologies utilizing stable transfected insectcells are also useful and are also desirably used in the presentinvention for VLP production. See, e.g., Pfeifer (1998) “Expression ofheterologous proteins in stable insect cell culture” Curr OpinBiotechnol. 9:518-21; Farrell et al. (1998) “High-level expression ofsecreted glycoproteins in transformed lepidopteran insect cells using anovel expression vector.” Biotechnol Bioeng 60 (6): 656-63; Lubiniecki,Cytotechnology (1998) 28:139-145; Murphy, et al., Curr. Prot. Mol. Biol.(1997) 16.9.1-16.9.10; McCarrol et al., Curr. Op. Biotech. (1997)8:590-594, Hegedus et al., Gene (1998) 207:241-249; Pfeifer et al.(1997) “Baculovirus immediate-early promoter-mediated expression of theZeocin resistance gene for use as a dominant selectable marker indipteran and lepidopteran insect cell lines.” Gene. 188:183-90;McLachlin and Miller (1997) “Stable transformation of insect cells tocoexpress a rapidly selectable marker gene and an inhibitor ofapoptosis” In Vitro Cell Dev Biol Anim. 33:575-9; McCarroll and King(1997) “Stable insect cell cultures for recombinant protein production”Curr Opin Biotechnol. 8:590-4; Vaughn, et al., (1997) In Vitro Cell Dev.Biol. 33:479-482; Jarvis et al. (1996) “Immediate-early baculovirusvectors for foreign gene expression in transformed or infected insectcells” Protein Expr Purif. 8:191-203; Schlaeger, (1996) Cytotechnology20:57-70; and Eaton, J. Chrom. A (1995) 705:105-114; Davies, Curr. Op.Biotech. (1995) 6:543-547, Potter, et al., Env. Health Pers. (1995)103:7-8, Poul, et al. Eur. J. Imm. (1995) 25:2005-2009; Bernard et al.,Cytotechnology (1994) 15 (1-3):139-144; Jarvis et al. (1995) “Continuousforeign gene expression in transformed lepidopteran insect cells.”Methods Mol Biol. 39:187-202; Lower, Cytotechnology (1995) 18:15-20;Hink et al., Biotech. Prog. (1991) 7:9-14; Jarvis et al. (1990) “Use ofearly baculovirus promoters for continuous expression and efficientprocessing of foreign gene products in stably transformed lepidopterancells.” Biotechnology 8:950-5; and Duane, et al., J. Tiss. Cult. Meth.(1989) 12 (1):13-16.

VLPs that Include Heterologous Domains

As discussed above, one feature of the invention is the insertion ofheterologous coding sequences into the RNA2 gene of a nodavirus at sitesthat do not interfere with capsid assembly. For example, in FHV,relevant sites for insertion of heterologous sequences include aminoacid residues 207-208 and 265-267 of the FHV coat protein.

The choice of which heterologous sequence to incorporate into the coatprotein depends on the application. In general, recombinant VLPs thatcomprise heterologous polypeptide sequences can be used either as amolecular decoy (antitoxin) or as a vaccine platform for binding andpresenting an immunogen.

When used as a molecular decoy, the VLP can include any of a variety ofanti-toxin moieties. For example, when the primary manifestations of adisease are caused by a microbial toxin, a corresponding antitoxin, ifadministered in time, can have a pronounced prophylactic or curativeeffect. Common bacterial toxins include botulism toxin, diphtheriatoxin, anthrax toxin, tetanus toxin and many others. Anti-toxincomponents that can be incorporated into the VLPs of the invention bindthese toxins in a patient, preventing their usual mode of action in thepatient. For example, as detailed in the examples herein, two anthraxtoxin receptors, widely distributed on human cells, have beenidentified: anthrax toxin receptor/tumor endothelial marker 8 (ANTXR1)⁷and capillary morphogenesis gene 2 (ANTXR2)⁸. Although both receptorsbind anthrax PA through a 200 amino acid extracellular von Willebrandfactor A (VWA) domain, the VWA domain of ANTXR2 has a 1000-fold higherbinding affinity for PA than the VWA domain of ANTXR1. Thus, VLPscomprising many copies (for FHV, 180 copies, or one per capsid proteinof the VLP), of the VWA domain of ANTXR2 acts as a potent anthraxantitoxin.

While this example serves to illustrate the invention, the example isnot limiting. The specific interactions between a variety of toxins andhost receptors are known and can similarly be used to select appropriateanti-toxin moieties for incorporation into VLPs. The major symptomsassociated, e.g., with disease caused by Corynebacterium diphtheriae(diphtheria), Bordetella pertussis (whooping cough), Vibrio cholerae(cholera), Bacillus anthracis (anthrax), Clostridium botulinum(botulism), Clostridium tetani (tetanus), and enterohemorrhagicEscherichia coli (bloody diarrhea and hemolytic uremic syndrome) aretoxin mediated, and the toxins and modes of action for these diseasesare all known (for additional examples, see, e.g.,drlera(dot)com/bacterial_diseases/protein_toxins(dot)htm#table on theworld wide web). For example, diptheria toxin binds to theheparin-binding epidermal growth factor HB-EGF receptor on susceptiblecells and enters by receptor-mediated endocytosis (seetextbookofbacteriology(dot)net/diphtheria(dot)html). By incorporatingthe ditheria toxin binding site of the HB-EGF receptor into a VLP of theinvention, e.g., at the sites noted in the examples herein relating toanthrax applications, an antitoxin against diptheria toxin is provided.Similarly, botulism toxin (botulinum toxin) binds irreversibly toreceptors on unmyelinated presynaptic membranes. This binding ismediated by a Carboxy (C) terminal of botulinum toxin heavy chain (inits native state, this heavy chain is associated with a light chain).Gangliosides with more than one neuraminic (sialic) acid, e.g. GT1b bindto the toxin. By incorporating glycoprotein components that comprisegangliosides into the VLP, a botulinum antitoxin is produced. Suchglycoproteins have been produced, see e.g., Hashimoto et al. (1998) “AStreptavidin-Based Neoglycoprotein Carrying More Than 140 GT1bOligosaccharides: Quantitative Estimation of the Binding Specificity ofMurine Sialoadhesin Expressed on CHO Cells” J. Biochem, 123 (3):468-478.

Decorating VLPs with Bound Heterologous Immunogens to Produce Vaccines

Binding Heterologous Immunogens to Heterologous Domains on VLPs

Heterologous polypeptide domains can be recombinantly fused with thecapsid protein of the relevant VLP e.g., as an in frame fusion domain,or as a chemically coupled domain to such a fusion to permit binding ofone or more immunogen to the VLP. The resulting immunogen decorated VLPscan be used as vaccines against the relevant immunogen. As described inthe examples herein, the heterologous domain can be one that binds tothe relevant immunogen directly, e.g., the VWA domain of ANTXR2, whichbinds Anthrax PA.

However, any high affinity interaction between a polypeptide domain anda binding partner can be utilized, e.g., by recombinantly or chemicallycoupling the binding partner (as an “adaptor” or “linker”) to theimmunogen, and then binding the resulting immunogen fusion to a VLP thatbinds the immunogen fusion. A large number of such cognate bindingpartners are known in the art and can be adapted to the practice of thepresent invention by being incorporated as VLP domains/immunogen fusionelements. For examples of binding partners, see, e.g.: Nilsson et al.(1997) “Affinity fusion strategies for detection, purification, andimmobilization of recombinant proteins” Protein Expression andPurification 11: 1-16; and Terpe et al. (2003) “Overview of tag proteinfusions: From molecular and biochemical fundamentals to commercialsystems” Applied Microbiology and Biotechnology 60:523-533, andreferences therein). Tags that can be used to couple the VLP to theimmunogen include, but are not limited to, a polyhistidine tag (e.g., aHis-6, His-8, or His-10 tag) that binds divalent cations (e.g., Ni²⁺), abiotin moiety (e.g., on an in vivo biotinylated polypeptide sequence ofthe VLP or immunogen) that binds avidin (present in the immunogen orVLP), a GST (glutathione S-transferase) sequence that binds glutathione,an S tag that binds S protein, an antigen that binds an antibody ordomain or fragment thereof (including, e.g., T7, myc, FLAG, and B tagsthat bind corresponding antibodies), a FLASH Tag (a high affinity tagthat couples to specific arsenic based moieties), a toxin receptor orreceptor domain that binds to the toxin, protein A or a derivativethereof (e.g., Z) that binds IgG, maltose-binding protein (MBP) thatbinds amylose, an albumin-binding protein that binds albumin, a chitinbinding domain that binds chitin, a calmodulin binding peptide thatbinds calmodulin, a cellulose binding domain that binds cellulose, etc.Another exemplary coupling partner that can be used to couple the VLP tothe immunogen is a SNAP-tag. The SNAP-tag is an approximately 20 kDaversion of a protein O⁶-alkylguanine-DNA alkyltransferase which has asingle reactive cysteine with a very high affinity for guaninesalkylated at the O⁶-position.

Generally speaking, the invention optionally includes the use of suchuniversal “adaptor” or “linker” domains in the immunogen of interest tofacilitate binding to a VLP. As noted, a variety of immunogens and theirbinding domains are known. Once a recombinant VLP is produced thatincludes a binding domain of interest, this becomes a useful platformfor coupling one or more different immunogens to the VLP. Thus, the VLPsherein that include the VWA domain of ANTXR2, which binds Anthrax PA canbe used as a general vaccine platform for the delivery of essentiallyany immunogen of interest. That is, an immunogen of interest is fused tothe portion of PA that is bound by a VWA domain (see Example 2 hereinfor details). The PA adaptor/linker binds the immunogen of interest tothe VLP. Such immunogens of interest include any of those noted herein,including ricin toxin and botulinum toxin.

This approach is also useful for making multivalent vaccinecompositions, e.g., by binding one or more VLP to one or more immunogenthrough a universal adaptor/linker that recognizes the VLP. Thus, aplurality of immunogen fusions that include an adaptor domain and animmunogen domain of interest can be bound to one or more VLP and thenadministered as a multivalent vaccine.

In any case, a recombinant VLP that can bind the immunogen of interestcan, typically, bind many copies of the immunogen. This is because theVLP can be made up of, e.g., 180 copies of the capsid protein, with eachcopy comprising an immunogen binding sequence. The precise number ofimmunogens that can decorate the VLP will vary, depending on the stericproperties of the immunogen. For example, the VLP will typically be ableto bind 10 or more copies, generally 50 or more copies, often 100 ormore copies, and sometimes up to 120 or more copies, and occasionally upto about 180 copies. In the case of anthrax PA binding to a VWA-VLP,described extensively herein, up to about 120 copies of PA can bind tothe VLP.

The immunogen and the recombinant VLP can be co-expressed in a singlecell, or the recombinant VLP and immunogen can be separately expressed.All references described regarding recombinant production and expressionof VLPs apply equally to expression and production of the immunogens.Methods of isolating VLPs are described above. Typical biomoleculepurification methods can similarly be used to isolate recombinantimmunogen, which can then be used to decorate purified recombinant VLPs.For example, protein isolation methods for isolating polypeptide-basedimmunogens are available, e.g., those set forth in the references notedabove in the context of VLP isolation.

Any of the examples above relating to VLPs comprising heterologousdomains can also equally relate to decoration of the VLP. That is,common bacterial toxins to be used to decorate the VLPs can includeanthrax toxin, botulism toxin, diphtheria toxin, tetanus toxin and manyothers. These can be coupled to the VLP either by binding to theirrespective antitoxin (incorporated into the VLP as noted above), or,e.g., by coupling through a standard heterologous binding partnerinteraction as noted herein.

The VLPs of the invention can display essentially any immunogen ofinterest, including immunogens that are not necessarily toxins. Forexample, VLPs can incorporate (or can be decorated with) relevantvaccine components related to significant vaccine targets such asbubonic plague. In this example, Yersinia pestis is the Gram-negativebacterium of the family Enterobacteriaceae responsible for bubonicplague (including the “black death”, likely caused by Yersinia pestisbiovar Medievalis). See, e.g., Camiel and Hinnebusch (eds.) (2004)Yersinia: Molecular and Cellular Biology Horizon Bioscience, InstitutPasteur, Paris, France and National Institutes of Health, Hamilton, USA;Collins F M (1996). “Pasteurella, Yersinia, and Francisella.” In:Barron's Medical Microbiology (Barron S et al, eds.), 4th ed. Y. pestisinfection can also cause pneumonic and septicemic plague. See also, RyanK J; Ray C G (editors) (2004). Sherris Medical Microbiology, 4th ed.,McGraw Hill, pp. 484-8; Parkhill et al. “Genome sequence of Yersiniapestis, the causative agent of plague” Nature 413: 523-527.Pathogenicity of Yersinia pestis is mediated by two anti-phagocyticantigens, known as F1 and VW, both of which are important for virulence.Collins F M (1996), above. Immunity (natural or induced) is achieved bythe production of antibodies against F1 and VW antigens; antibodiesagainst F1 and VW induce phagocytosis by neutrophils. Salyers and Whitt(2002). Bacterial Pathogenesis: A Molecular Approach, 2nd ed., ASMPress. 207-12.

The F1 antigen is one preferred vaccine target. F1 is an outer membranecapsular protein of Y. pestis. The immunogenicity of whole F1 and ofvarious peptide sequences, e.g, predicted to possess B (three sequences,B1, B2 and B3) and T (two sequences, T1 and T2) cell determinants havebeen studied, as have chimeras made between B and T structures. See,e.g., Sabhnani (2003) “Developing subunit immunogens using B and T cellepitopes and their constructs derived from the F1 antigen of Yersiniapestis using novel delivery vehicles.” FEMS Immunol Med Microbiol. 38(3):215-29; Grosfeld et al. (2003) “Effective Protective Immunity toYersinia pestis Infection Conferred by DNA Vaccine Coding forDerivatives of the F1 Capsular Antigen” Infect Immun. 71 (1): 374-383.Virulent and potentially virulent cells of Yersinia pestis producevirulence or V and W antigens (VW(+)); these antigens are also preferredvaccine targets for VLP decoration. For example, antibodies against Vprotein protect against Yersinia pestis infection and can be used todecorate VLPs of the invention. See also, e.g., Pullen et al. (1998)“Analysis of the Yersinia pestis V Protein for the Presence of LinearAntibody Epitopes” Infect Immun. 66 (2): 521-527. Vaccines thatincorporate both V and F1 elements are also in development. See also,Elvin and Williamson (2000) “The F1 and V subunit vaccine protectsagainst plague in the absence of IL-4 driven immune responses” Microb.Pathog. 29:223-230; see also Anisimov et al. (2004) “IntraspecificDiversity of Yersinia pestis” Clinical Microbiology Reviews 17 (2):434-464; Williamson (2001) “Plague vaccine research and Development”Journal of Applied Microbiology 91:606-608.

Accordingly, in one embodiment of the invention, VLPs are decorated withF1 and/or V protein, or subsequences thereof, using any of theapproaches already noted for binding the F1 and/or V proteins or peptidesubsequences thereof to the VLP. F1 and/or V proteins, or portionsthereof (e.g., for F1, B1, B2, B3 and/or T1 and/or T2), can also berecombinantly incorporated into the VLPs of the invention, at one ormore of the loop sites noted herein. In either case, the resulting VLPsare used as an immunogen to raise protective antibodies against F1and/or V protein. VLPs as Anthrax Vaccines

In one particularly preferred aspect, VLPs of the invention aredecorated with anthrax toxin components (typically PA, or polypeptidesequences derived from PA). Multivalent display of the ANTXR2 VWA domainon the surface of the icosahedral insect nodavirus FHV VLP was shown toinduce a potent immune response against LeTx when coated with PA. Thisimmune response was neutralizing in vitro and protected animals againstLeTx challenge following a single administration without adjuvant.

While the examples below refer to particular ANTRX2 receptorpolypeptides and PA sequences, one of skill will recognize that theinvention equally encompasses allelic variants of particular sequences.Further, the present invention is not limited to these particularexamples. Recombinant VLPs can, for example, include larger segments ofthe ANTRX2 receptor polypeptide than the VWA sequence, and can alsoinclude, e.g., purification components (e.g., VLP purification tags thatinclude any of the binding partners noted above). In addition,conservative variations of the ANTRX2 receptor polypeptide (or VWAcomponent thereof) can readily be substituted. Preferably, any suchconservative substitutions of the VWA domain will still be bound by therelevant immunogen, e.g., a PA domain (or conservative variant thereof)will preferably bind to the relevant recombinant VLP. This can be testedfor in a routine VLP-immunogen binding assay, e.g., as taught in theexamples herein.

“Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid or polypeptide are those thatencode identical or essentially identical amino acid sequences. One ofskill will recognize that individual substitutions, deletions oradditions which alter, add or delete a single amino acid or a smallpercentage of amino acids (typically less than 5%, more typically lessthan 4%, 2% or 1%) in an encoded sequence are “conservatively modifiedvariations” where the alterations result in the deletion of an aminoacid, addition of an amino acid, or substitution of an amino acid with achemically similar amino acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Table A sets forth six groups whichcontain amino acids that are “conservative substitutions” for oneanother.

TABLE A Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

Thus, “conservatively substituted variations” of a given polypeptidesequence of the present invention (e.g., ANTRX2 receptor, anthrax PA,etc,) include substitutions of a small percentage, typically less than5%, more typically less than 2% or 1%, of the amino acids of thepolypeptide sequence, with a conservatively selected amino acid of thesame conservative substitution group.

The addition or deletion of sequences that do not alter the encodedactivity of a nucleic acid molecule, such as the addition or deletion ofa non-functional sequence, is a conservative variation of the basicnucleic acid or polypeptide.

One of skill will appreciate that many conservative variations of thenucleic acid constructs that are disclosed yield a functionallyidentical construct. For example, owing to the degeneracy of the geneticcode, “silent substitutions” (i.e., substitutions in a nucleic acidsequence that do not result in an alteration in an encoded polypeptide)are an implied feature of every nucleic acid sequence that encodes anamino acid. Similarly, “conservative amino acid substitutions,” in oneor a few amino acids in an amino acid sequence are substituted withdifferent amino acids with highly similar properties, are also readilyidentified as being highly similar to a disclosed construct. Suchconservative variations of each relevant sequence are a feature of thepresent invention. Further details regarding sequence variations andmethods of mutagenesis are found below.

Sequence Variations

A variety of ANTRX2 polypeptides and nucleic acids are known, includingthose found in GenBank (see, Definitions section herein).

Accordingly, a number of polypeptides and coding nucleic acids aredescribed herein by sequence (See, e.g., the accession information andExamples sections below). These polypeptides and coding nucleic acidscan be modified, e.g., by mutation as described herein, or simply byartificial synthesis of a desired variant. Several types of examplevariants are described further below.

Silent Variations

Due to the degeneracy of the genetic code, any of a variety of nucleicacids sequences encoding polypeptides of the invention are optionallyproduced, some which can bear various levels of sequence identity to thenucleic acids or polypeptides in the Examples below. The followingprovides a typical codon table specifying the genetic code, found inmany biology and biochemistry texts.

TABLE B Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gin QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

The codon table shows that many amino acids are encoded by more than onecodon. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the invention where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

Such “silent variations” are one species of “conservatively modifiedvariations”, discussed above. One of skill will recognize that eachcodon in a nucleic acid (except ATG, which is ordinarily the only codonfor methionine) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention, therefore, explicitly provides eachand every possible variation of a nucleic acid sequence encoding apolypeptide of the invention that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code (e.g., as setforth in Table B, or as is commonly available in the art) as applied tothe nucleic acid sequence encoding a polypeptide of the invention. Allsuch variations of every nucleic acid herein are specifically providedand described by consideration of the sequence in combination with thegenetic code. One of skill is fully able to make these silentsubstitutions using the methods herein.

Mutational Derivatives

The nucleic acid and protein sequences herein can be mutated by standardmethods, e.g., to improve binding between the immunogen and VLP, toimprove stability or half-life, or the like. Additional information onmutation formats is found in Sambrook and Ausubel, as well as in PCRProtocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis). The followingpublications and references provide additional detail on mutationformats: Arnold, Protein engineering for unusual environments, CurrentOpinion in Biotechnology 4:450-455 (1993); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Botstein & Shortle, Strategies and applications of in vitromutagenesis, Science 229:1193-1201 (1985); Carter et al., Improvedoligonucleotide site-directed mutagenesis using M13 vectors, Nucl. AcidsRes. 13: 4431-4443 (1985); Carter, Site-directed mutagenesis, Biochem.J. 237:1-7 (1986); Carter, Improved oligonucleotide-directed mutagenesisusing M13 vectors, Methods in Enzymol. 154: 382-403 (1987); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Eghtedarzadeh & Henikoff,Use of oligonucleotides to generate large deletions, Nucl. Acids Res.14: 5115 (1986); Fritz et al., Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro, Nucl. Acids Res. 16: 6987-6999 (1988); Grundström et al.,Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ genesynthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Kunkel, The efficiencyof oligonucleotide directed mutagenesis, in Nucleic Acids & MolecularBiology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag,Berlin)) (1987); Kunkel, Rapid and efficient site-specific mutagenesiswithout phenotypic selection, Proc. Natl. Acad. Sci. USA 82:488-492(1985); Kunkel et al., Rapid and efficient site-specific mutagenesiswithout phenotypic selection, Methods in Enzymol. 154, 367-382 (1987);Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Point Mismatch Repair, Cell 38:879-887 (1984);Kramer et al., Improved enzymatic in vitro reactions in the gappedduplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Ling et al., Approaches toDNA mutagenesis: an overview, Anal Biochem. 254 (2): 157-178 (1997);Lorimer and Pastan Nucleic Acids Res. 23, 3067-8 (1995); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakamar and Khorana, Total synthesis and expression of a genefor the a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Sayers etal., Y-T Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis, Nucl. Acids Res. 16:791-802 (1988); Sayers et al., Strandspecific cleavage of phosphorothioate-containing DNA by reaction withrestriction endonucleases in the presence of ethidium bromide, (1988)Nucl. Acids Res. 16: 803-814; Sieber, et al., Nature Biotechnology,19:456-460 (2001); Smith, In vitro mutagenesis, Ann. Rev. Genet.19:423-462 (1985); Methods in Enzymol. 100: 468-500 (1983); Methods inEnzymol. 154: 329-350 (1987); Stemmer, Nature 370, 389-91 (1994); Tayloret al., The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985);Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8787 (1985); Wells et al., Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Zoller &Smith, Oligonucleotide-directed mutagenesis using M13-derived vectors:an efficient and general procedure for the production of point mutationsin any DNA fragment, Nucleic Acids Res. 10:6487-6500 (1982); Zoller &Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors, Methods in Enzymol. 100:468-500 (1983); and Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987). Additional details on many of the abovemethods can be found in Methods in Enzymology Volume 154, which alsodescribes useful controls for trouble-shooting problems with variousmutagenesis methods.

In addition, serum half-life and other properties of VLPs and immunogenscan be modulated using mutagenesis, or by other well known methods, suchas by the addition of PEG or other protective (e.g., saccharide)moieties to the enzymes.

Pharmaceutical Compositions and Administration to Patients

Antitoxins and immunogenic compositions of the invention can beformulated into pharmaceutical compositions. These compositions maycomprise, in addition to one or more VLP (or decorated VLP), anavailable pharmaceutically acceptable excipient, carrier, buffer,stabilizer or the like. Such materials should typically be non-toxic andshould not interfere with the efficacy of the active ingredient. Theprecise nature of the carrier or other material depends on the route ofadministration, e.g., whether administration is via oral, rectal,intravenous, cutaneous, subcutaneous, nasal, intramuscular,intraperitoneal or other routes. The route is chosen to put thecomposition into contact either with the immune system, or with the siteof toxin production/activity.

For example, pharmaceutical compositions for oral administration may bein tablet, capsule, powder or liquid form. A tablet may include a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

For intravenous, cutaneous or subcutaneous injection, or localinjection, e.g., at the site of an affliction (e.g., a skin lesion), theactive ingredient will be in the form of a parenterally acceptableaqueous solution which has suitable pH, isotonicity and stability. Thoseof skill in the art are able to prepare suitable solutions using, forexample, isotonic vehicles such as sodium chloride injection, Ringer'sinjection, lactated Ringer's injection, or the like. Preservatives,stabilizers, buffers, antioxidants and/or other additives are alsooptionally included, as required.

Whether it is a VLP antitoxin or immunogenic composition, that is to begiven to an individual, administration is preferably in a“prophylactically effective amount” (e.g., enough to prevent orameliorate the effects of a disease, e.g., anthrax infection) or a“therapeutically effective amount” (prophylaxis optionally also can beconsidered therapy), this being an amount sufficient to show a benefitto the individual (e.g., enough to prevent death from the relevanttoxin, or to induce a protective immune response to the disease agent).The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Prescription of treatment, e.g. decisions on dosage etc, iswithin the responsibility of general practitioners and other medicaldoctors, and typically takes account of the disorder to be treated, thecondition of the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found, e.g., in thecurrent edition of Remington's e.g., Remington: The Science and Practiceof Pharmacy, Twenty First Edition (2005).

The compositions may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Thus, in the treatment of anthrax, the VLP,etc., can be administered in combination with other available therapies.

The compositions can be administered to human patients, or to non-humanveterinary patients. For example, anthrax infects sheep, cattle andother livestock animals, as well as humans, and the compositions of theinvention can be used to treat livestock as well as human patients.

Additional Details for Administering Antitoxins

Antitoxins are preferably administered as soon as possible afterexposure to a toxin. The route of administration can vary depending onthe application. Many toxins are active in the circulatory system,making i.v. or i.p. delivery especially useful. However, many toxinsalso can act e.g., in the digestive system (e.g., botulinum toxins),making oral delivery appropriate in those instances, or in the nervoussystem (e.g., diphtheria toxin) making CSF, intracranial or spinaladministration appropriate.

Additional Details for Administering VLP Vaccines and Making Antibodies

VLP vaccines can be administered in a manner designed to elicit animmune response. This can typically be achieved by simple injection(s.c., i.m., etc.). Optionally, a decorated VLP vaccine can beadministered in conjunction with an adjuvant designed to boost an immuneresponse, e.g., depending on the host species, including, but notlimited to, Freund's (complete or incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Decorated VLP vaccines, in addition to being useful as protective agents(vaccines), are also useful in the production of antibodies against adisplayed immunogen. Such antibodies are useful as labeling reagents todetect the immunogen, e.g., to detect or monitor infection in a patientfor diagnostic or prognostic purposes, or for in situ labeling inclinical or laboratory applications. In addition, such antibodies act asantitoxins against the immunogen at issue. Thus, antibodies can beraised in one host, isolated by standard methods, and injected into apatient as an antitoxin.

As used herein, the term “antibody” includes, but is not limited to,polyclonal antibodies, monoclonal antibodies, humanized or chimericantibodies and biologically functional antibody fragments, which arethose fragments sufficient for binding of the antibody fragment to theprotein.

For the production of antibodies to a decorated VLP, various hostanimals may be immunized by injection with the VLP. Such host animalsmay include human patients, livestock animals (e.g., sheep, cattle,hogs, horses, etc.), or laboratory animals (e.g., rabbits, mice orrats).

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with the VLP. Forthe production of polyclonal antibodies, host animals, such as thosedescribed above, may be immunized by injection with the VLP, e.g.,supplemented with adjuvants as also described above.

Monoclonal antibodies (mAbs), which are homogeneous populations ofantibodies to a particular VLP antigen, may be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein (Nature 256:495-497, 1975;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Nat'l.Acad. Sci. USA 80:2026-2030, 1983), and the EBV-hybridoma technique(Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96, 1985). Such antibodies may be of any immunoglobulinclass, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

For details regarding antibody production and their use in assays, insitu antigen detection, or the like, see Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork; Harlow and Lane (1998) Using Antibodies: A Laboratory Manual ColdSpring Harbor Lab Press ISBN-10: 0879695447; Schelling (2002) MonoclonalAntibody Protocols ISBN: 0896036553, Humana Press; Welschof and Krauss(2002) Recombinant Antibodies for Cancer Therapy: Reviews and Protocols(Methods in Molecular Biology) ISBN: 0896039188 Humana Press; Albitar(2007) Monoclonal Antibodies: Methods and Protocols (Methods inMolecular Biology) ISBN: 1588295672 Humana Press.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not to be imputed to any related or unrelated case, e.g., to anycommonly owned patent or application. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the present invention, the preferred materialsand methods are described herein. Accordingly, the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

An “antitoxin” is a composition that blocks an activity of a toxin in anorganism. Common antitoxins bind to the toxin, blocking the toxin'susual mode of operation. Antitoxins can include molecular decoys such astoxin receptor molecules or portions thereof that bind to the toxin,and/or antibodies previously raised against the toxin.

An “anthrax antitoxin” is an antitoxin that ameliorates an effect of ananthrax toxin. For example, a recombinant VLP of the invention caninclude a VWA domain of an ANTRX2 receptor, which acts as a moleculardecoy by binding PA. This results in protection against effects of theanthrax toxin.

The term “anthrax toxin” refers to any component or product of theanthrax toxin. That is, the toxic effects of anthrax are predominantlydue to an AB-type toxin made up of a single receptor-binding B subunitand two enzymatic A subunits. The A subunits are edema factor (EF, 89kD), an adenylate cyclase that raises intracellular cAMP levels, andlethal factor (LF, 90 kD), a zinc protease that cleavesmitogen-activated protein kinase kinases. The receptor binding B subunitis protective antigen (PA), which is initially synthesized as an 83 kDprecursor. Upon receptor binding, PA83 is cleaved by furin into a 63 kDproduct, which forms heptamers that bind EF to form edema toxin (EdTx)and LF to form lethal toxin (LeTx). Any or all of these are encompassedby the term “anthrax toxin.” In addition, molecules derived from awild-type anthrax toxin, e.g., through mutagenesis or recombinantmethods, are also included within the term.

An “anthrax protective antigen” (PA) is the portion of an anthrax toxinthat recognizes an anthrax receptor (e.g., anthrax toxin receptor/tumorendothelial marker 8 (ANTXR1) or capillary morphogenesis gene 2(ANTXR2)). Both the 83 kD precursor and the 63 kD furin cleavage productare optionally included within the term, as is the heptameric formthereof. In addition, molecules derived from a wild-type anthrax PA,e.g., through mutagenesis or recombinant methods, are also includedwithin the term.

A “capillary morphogenesis gene 2” (ANTRX2) gene encodes an ANTRX2protein which is a receptor protein that mediates Bacillus anthraciskilling of macrophages following spore challenge. A variety of allelesand synonyms for ANTRX2 are known in the art. The universal proteinknowledge database (UniProt) has several accession numbers for ANTRX2proteins, including P58335, Q4W5H6 and Q32Q26 (GenBank accession no.AY23345). NCBI has an accession number for the coding gene (EnterezGeneID: 118429). Synonyms for the protein include anthrax toxin receptor2 precursor, Capillary morphogenesis gene 2 protein, CMG2, CMG-2,FLJ31074, ISH, JHF, MGC111533, and MGC45856. In the present invention,the terms for the polypeptide and protein include all allelic variantsof the given polypeptides and genes at the accession numbers above, aswell as derivative variants thereof, particularly those that can bindPA. ANTRX2 typically binds to the protective antigen (PA) of Bacillusanthracis in a divalent cation-dependent manner, with the followingpreference: calcium>manganese>magnesium>zinc. Binding of PA leads toheptamerization of the receptor-PA complex. The gene and correspondingprotein are well characterized, binding laminin, and collagen type IV.

Additional details regarding ANTRX2 proteins and nucleic acids can befound in Genebank at AAI07877 (human) NP_(—)598499 487 aa linear anthraxtoxin receptor 2 [Mus musculus]; NP_(—)477520, anthrax toxin receptor 2[Homo sapiens] gi|50513243|ref|NP_(—)477520.2|[50513243]; P58335 Anthraxtoxin receptor 2 precursor (Capillary morphogenesis gene 2 protein)(CMG-2); AAH76595 cDNA clone, Anthrax toxin receptor 2 [Mus musculus]gi|49901393|gb|AAH76595.1|[49901393]; ABD74633 capillary morphogenesisprotein 2A [Danio rerio]; NM_(—)058172 Homo sapiens anthrax toxinreceptor 2 (ANTXR2), mRNA gi|68342041|ref|NM_(—)058172.3|[68342041];NM_(—)133738 Mus musculus anthrax toxin receptor 2 (Antxr2), mRNAgi|50355938|ref|NM_(—)133738.1|[50355938]; BC107876 cDNA clone, Homosapiens anthrax toxin receptor 2, mRNA (cDNA clone MGC:111533IMAGE:6108432), complete cds gi|79154031|gb|BC107876.1|[79154031];BC076595 cDNA clone, Mus musculus anthrax toxin receptor 2, mRNA (cDNAclone MGC: 100142 IMAGE:30649897), complete cdsgi|49901392|gb|BC076595.1|[49901392]; DQ415957 Danio rerio capillarymorphogenesis protein 2A (cmg2a) mRNA, complete cds;gi|89513612|gb|DQ415957.1|[89513612]; BC034001 Homo sapiens anthraxtoxin receptor 2, mRNA (cDNA clone IMAGE:4894326), complete cdsgi|21708157|gb|BC034001.1|[21708157]; DV770783 ILLUMIGEN_MCQ_(—)70414Katze_MMOV Macaca mulatta cDNA clone IBIUW:40677 5-similar to Bases 26to 766 highly similar to human ANTXR2 (Hs.162963), mRNA sequencegi|82614725|gb|DV770783.1|[82614725]; BC123757 cDNA clone, Bos taurussimilar to Anthrax toxin receptor 2 precursor (Capillary morphogenesisgene-2 protein) (CMG-2), mRNA (cDNA clone MGC:143297 IMAGE:8231847),complete cds gi|115305013|gb|BC123757.1|[115305013]; NM_(—)001076826 Bostaurus similar to Anthrax toxin receptor 2 precursor (Capillarymorphogenesis gene-2 protein) (CMG-2) (MGC143297), mRNAgi|116003874|ref|NM_(—)001076826.1|[116003874]; NM_(—)001044709 Daniorerio similar to Anthrax toxin receptor 2 precursor (Capillarymorphogenesis protein-2) (CMG-2) (LOC557239), mRNAgi|113374124|ref|NM_(—)001044709.1|[113374124]; NM_(—)058172 Homosapiens anthrax toxin receptor 2 (ANTXR2), mRNAgi|68342041|ref|NM_(—)058172.3|[68342041]; NM_(—)133738 Mus musculusanthrax toxin receptor 2 (Antxr2), mRNAgi|50355938|ref|NM_(—)133738.1|[50355938]; BC003908 Mus musculus anthraxtoxin receptor 2, mRNA (cDNA clone IMAGE:3484366), partial cdsgi|13278123|gb|BC003908.1|[13278123]; XM_(—)680988 Danio rerio similarto Anthrax toxin receptor 2 precursor (Capillary morphogenesisprotein-2) (CMG-2) (LOC557845), mRNA; AAI23758 cDNA clone, Similar toAnthrax toxin receptor 2 precursor (Capillary morphogenesis gene-2protein) (CMG-2) [Bos taurus]; XP_(—)686080 similar to Anthrax toxinreceptor 2 precursor (Capillary morphogenesis protein-2) (CMG-2) [Daniorerio]; AAK77222 capillary morphogenesis protein-2 [Homo sapiens].

An “extracellular von Willebrand Factor A” (VWA) domain is a polypeptidedomain that displays structural similarity to the VWA domain originallydescribed in the blood coagulation protein von Willebrand factor (VWF).VWA domains fold into a compact three-layered para-Rossmann type fold,consisting of seven helices surrounding a core of five parallelbeta-strands and one short antiparallel beta-strand. VWAs are a wellstudied protein domain typically involved in cell adhesion,extracellular matrix proteins, and in integrin receptors. The crystalstructure of the VWA domain from ANTRX2 (as well as many other proteins)has been solved. See, Lacy et al. (2004) Crystal structure of the vonWillebrand factor A domain of human capillary morphogenesis protein 2:An anthrax toxin receptor Proc Natl Acad Sci USA. 101 (17): 6367-6372(Published online 2004 Apr. 12. doi: 10.1073/pnas.0401506101).

A domain is “heterologous” to a specified polypeptide when it is derivedfrom a polypeptide that is different from the specified polypeptide. Aheterologous toxin binding domain of a VLP is a polypeptide domain thatis heterologous to the capsid protein of the VLP and that binds a toxinmoiety. A heterologous immunogen binding domain of a VLP is a domainthat is heterologous to the capsid protein of the VLP and that binds animmunogen. A heterologous toxin binding domain and a heterologousimmunogen binding domain can, in some embodiments, be the same domain.For example, the VWA domain of an ANTRX2 polypeptide binds anthrax toxin(PA), making it an antitoxin, as well as an immunogen binding domain forthe display of PA.

A “nodavirus-derived virus like particle” is a capsid that is made up ofcapsid proteins that are derived from a nodavirus. The capsid proteinscan be nodavirus capsid proteins, or can be recombinant proteins thatinclude sequences derived from such capsid proteins (or both). Ingeneral, a second polypeptide is “derived from” a first polypeptide whenthe second polypeptide (or coding nucleic acid thereof) is producedusing sequence information from the first polypeptide, or a codingnucleic acid thereof, or when the second polypeptide (or coding nucleicacid thereof) is produced from the first polypeptide (or coding nucleicacid thereof) by artificial, e.g., recombinant methods. For example,when the second polypeptide is made by mutating a nucleic acid encodingthe first polypeptide, and expressing the resulting mutated nucleicacid, the second polypeptide is said to be “derived from” the first.Similarly, when the second polypeptide is made using sequenceinformation from the first polypeptide, e.g., by mutating the sequenceof the first polypeptide in silico and then synthesizing, e.g., acorresponding nucleic acid that encodes the second polypeptide andexpressing it, the resulting second polypeptide is derived from thefirst polypeptide.

Virus Like Particles (VLPs) are also alternatively referred to as viralnanoparticles (VNs). The ANTRX2 VLP/VN herein denoted as including theextracellular von Willebrand Factor A (VWA) domain, e.g., domain I, arealternately denoted VNI (viral nanoparticles with I domain). Therefore,the uncomplexed form is alternatively denoted VNI and the form with PAbound is alternately denoted VNI-PA.

An amino acid residue in a protein “corresponds” to a given residue whenit occupies the same essential structural position within the protein asthe given residue. For example, a selected residue in a selectednodavirus protein other than FHV corresponds to residue 206 (or e.g.,264) of the FHV capsid protein when the selected residue occupies thesame essential spatial or other structural relationship to other aminoacids in the selected protein as residue 206 (or 264) does with respectto the other residues in the FHV capsid protein. Thus, if the selectedprotein is aligned for maximum homology with the FHV capsid protein, theposition in the aligned selected protein that aligns with FHV capsidresidue is said to correspond to it. Instead of a primary sequencealignment, a three dimensional structural alignment can also be used,e.g., where the structure of the selected protein is aligned for maximumcorrespondence with the FHV capsid protein and the overall structurescompared. In this case, an amino acid that occupies the same essentialposition a residue in the structural model is said to correspond to theresidue.

A “toxin” is a toxic substance, typically of biological origin. Avariety of toxins are made by bacteria (anthrax toxin, botulinum toxin,necrotizing toxin from Necrotizing fasciitis, etc.), as well as, e.g., avariety of jellyfish, snakes, insects and spiders. Toxins relevant tothe invention typically include polypeptides or polypeptide components.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. Accordingly, the following examples areoffered to illustrate, but not to limit, the claimed invention.

Example 1 A Viral Nanoparticle with Dual Function as an AnthraxAntitoxin and Vaccine

The recent use of Bacillus anthracis as a bioweapon has stimulated thesearch for novel antitoxins and vaccines that act rapidly and withminimal adverse effects. B. anthracis produces an AB-type toxin composedof the receptor-binding moiety protective antigen (PA) and the enzymaticmoieties edema factor and lethal factor. PA is a key target for bothantitoxin and vaccine development. We used the icosahedral insect virusFlock House virus as a platform to display 180 copies of the highaffinity, PA-binding von Willebrand A (VWA) domain of the ANTXR2cellular receptor. The chimeric virus-like particles (VLPs) correctlydisplayed the receptor VWA domain on their surface and inhibited lethaltoxin action in in vitro and in vivo models of anthrax intoxication.Moreover, VLPs complexed with PA elicited a potent toxin-neutralizingantibody response that protected rats from anthrax lethal toxinchallenge after a single immunization without adjuvant. This recombinantVLP platform represents a novel and highly effective dually-actingreagent for treatment and protection against anthrax.

To develop a reagent that functions both as an anthrax antitoxin and asa molecular scaffold for an efficient anthrax vaccine, we took advantageof an icosahedral virus platform that permits polyvalent display of theextracellular VWA domain of ANTXR2. This platform is based on FlockHouse virus (FHV), a non-enveloped, icosahedral (T=3) insect virus ofthe family nodaviridae [16]. The FHV capsid is composed of 180 subunitsof a single type of coat protein, and the icosahedral, solid shellencapsidates a bipartite, single-stranded RNA genome. The crystalstructure of FHV particles shows that the coat protein contains severalsurface-exposed loops that can be targeted for insertion of foreignproteins and peptides [17]. Here we report the synthesis and structuralcharacterization of FHV-VWA_(ANTXR2) chimeric particles and provideevidence for their efficacy as an anthrax toxin inhibitor in vitro andin vivo. In addition, we used the chimeric particles as a scaffold forthe multivalent display of PA and show that this complex functions as apotent vaccine against LeTx.

FHV-VWA_(ANTXR2) Chimeric Proteins Assemble into Virus-Like-Particles.

The VWA domain of ANTXR2 forms a compact structure that adopts aRossmann-like α/β-fold with a metal ion-dependent adhesion site motifthat is involved in PA binding [9,18]. The N and C termini of thisdomain, residues C39 and C218, respectively, are closely juxtaposedthereby permitting, in principle, genetic insertion into a loop on acarrier protein. Modeling studies of the FHV coat protein subunitindicated that two surface-exposed loops at amino acid positions 206 and264 would accommodate the 181 amino acid ANTXR2VWA domain withoutdisrupting coat protein assembly into virus-like particles (VLPs) (FIG.1A). Based on these predictions, two chimeric proteins were generated.In FHV-VWA_(ANTXR2) chimera 206, the VWA domain and a C-terminal twoamino acid linker (Ala-Glu) replaced FHV coat protein residues 207-208(FIG. 1B). In FHV-VWA_(ANTXR2) chimera 264, the VWA domain replaced FHVresidues 265-267. The chimeric proteins were expressed in Sf21 insectcells using recombinant baculovirus vectors. In this system, wildtype(wt) FHV coat protein forms VLPs whose high resolution structure isvirtually indistinguishable from that of native virions. However, VLPscontain random cellular RNA instead of the FHV genome and are thereforenot infectious [19].

Putative chimeric VLPs were purified from the cells by sucrose gradientcentrifugation and material sedimenting at a position similar to thatobserved for native virions was harvested and analyzed by SDS-PAGE. Asshown in FIG. 1C, both samples contained a major protein and a slowermigrating minor protein of the appropriate molecular weights (≈63 kD,the combined molecular weight of the 43 kD FHV coat protein and the 20kD ANTXR2 VWA domain). Since the FHV coat protein undergoes aspontaneous cleavage reaction after assembly of particles (FIG. 1B)[20], the minor protein likely represented the unprocessed precursorprotein, whereas the major protein represented the post-assemblycleavage product. Capsid proteins representing chimera 264 migrated moreslowly through the gel than those representing chimera 206, even thoughthe amino acid composition of the two polypeptides was virtuallyidentical. The reason for this differential behavior is not known butcould reflect subtle differences in denaturation of the proteins underSDS-PAGE conditions.

Electron microscopy of negatively stained samples confirmed the presenceof VLPs in the gradient-purified material (FIG. 1D). Compared to thesmooth exterior of native FHV virions, the surface of the chimericparticles was rough and distinct protrusions were visible. Theappearance of the particles suggested that they were filled with RNA, asstain did not penetrate the interior. This conclusion was supported bythe sedimentation rate of the VLPs, which was indistinguishable fromthat of wt FHV.

Structural Analysis of FHV-VWA_(ANTXR2) VLPS.

Electron cryomicroscopy and image reconstruction of the FHV-VWA_(ANTXR2)VLPs showed that, compared to wt FHV particles (FIG. 5C), both chimericparticles displayed additional density at higher radius (FIGS. 5A and5B), in agreement with the protrusions that were visible in negativelystained samples (FIG. 1D). To define the arrangement of the VWA domainson the surface of the chimeric particles, pseudoatomic models weregenerated by fitting the X-ray coordinates of the FHV coat proteinsubunit and the ANTXR2 VWA domain into the cryoEM density maps (FIGS. 3Aand 3B; 6A and 6B). The models revealed that in chimera 206 the VWAdomains were closely juxtaposed at the quasi three-fold axes. Two of thethree VWA domains in each asymmetric unit closely interacted with theirtwofold related counterparts, thereby creating an offset cluster of sixdomains. In contrast, the insertion site chosen for chimera 264 allowedfor wider spacing and more even distribution of the individual VWAdomains on the particle surface.

To investigate the accessibility of PA to the VWA domains, PA83 wascomputationally docked onto the VWA domains of the pseudoatomic modelsof chimeras 264 and 206 using the X-ray structure of PA63 complexed withthe ANTXR2 VWA domain as a guide [9,18]. It was evident that chimera 264could accommodate significantly more PA molecules than chimera 206 giventhe wider spacing of the VWA domains on this particle (FIGS. 3C and 3D;7A and 7B). Specifically, each subunit at the fivefold axes and three ofthe six subunits around the quasi-sixfold axes could bind PA83 withoutsteric interference, giving a total occupancy of 120 PA molecules perparticle. In contrast, due to the close juxtaposition of the VWA domainson chimera 206, a maximum occupancy of 60 PA molecules per particle waspredicted. These predictions were in close agreement with results frombiochemical analyses of complexes formed between the particles and PA83under saturating conditions. Specifically, gel electrophoresis combinedwith densitometric analysis showed that chimera 206 could bind anaverage of 90 PA83 ligands, whereas chimera 264 bound an average of 130PA83 ligands (FIGS. 8A and 4B). Together, these results were consistentwith the observation that a higher concentration of chimera 206 wasrequired to protect cells from intoxication with PA/LF_(N)-DTA (FIG. 2).

FHV-VWA_(ANTXR2) VLPS Protect Cultured Cells from Intoxication

The soluble, monomeric ANTXR2 VWA domain (sANTXR2), expressed andpurified from mammalian cells, was previously shown to effectively blockentry of lethal toxin into susceptible cells by competing with cellularANTXR2 for binding to PA [10]. PA has a very high binding affinity forsANTXR2 (K_(d)=170 pM) and dissociates extremely slowly from thisreceptor decoy (half-life of the complex is approximately 17 hours)[21]. We used the same approach to test the inhibitory activity ofFHV-VWA_(ANTXR2) VLPs. Namely, the assay employed CHO-K1 cells and amodified form of lethal toxin, PA/LF_(N)-DTA, in which the N terminalportion of LF was fused to the catalytic portion of diphtheria toxinA-chain [22]. This recombinant toxin efficiently kills CHO-K1 cellswithin 48 hours and uses the same PA-dependent entry mechanism as wt LF.The assay revealed that chimera 264 protected cells as efficiently assANTXR2, whereas a higher concentration of chimera 206 was required toachieve protection of the cells (FIG. 2). The corresponding IC₅₀ valuesfor sANTXR2 and chimera 264 were 19.70±0.87 nM and 18.50±0.36 nM,respectively, while the IC₅₀ was 32.71±0.61 nM for chimera 206. Thus,chimera 264 performed as well as the highly potent, monomeric sANTXR2inhibitor in this assay. To confirm the ability of the particles toneutralize native lethal toxin, a macrophage-based toxin neutralizationassay was performed with chimera 264. The assay revealed that theparticles protected RAW264.7 cells efficiently from a mixture of PA andwt lethal toxin and the measured IC₅₀ was 39.8+2.2 nM.

FHV-VWA_(ANTXR2) Chimera 264 Protects Rats from Lethal Toxin Challenge.

We next tested whether the chimeric particles were capable of protectingrats against lethal toxin challenge as was demonstrated previously forsANTXR2 [10]. In vivo experiments were only performed with chimera 264because it had shown higher potency in the cell intoxication assay (FIG.2). As a positive control, sANTXR2 was used in parallel. Male Fisher 344rats were inoculated intravenously with 5 minimal lethal doses (MLD) ofLeTx either in the presence or absence of chimera 264 or sANTXR2 aspreviously described [10]. As shown in Table 1, both chimera 264 andsANTXR2 completely protected the animals when used at a molar ratio of2:1 (ANTXR2:PA). Moreover, the animals did not exhibit any symptoms ofintoxication such as agitation, respiratory distress or hypoxia.Injection vehicle (PBS) or wt FHV, used as a negative control, had noprotective effect. While neither chimera 264 nor sANTXR2 were able toprotect rats when used at a tenfold lower concentration (molar ratio ofANTXR2:PA=0.2:1), they each caused a delay in the time to death comparedto the lethal toxin control (Table 1). The delay was notably longer forchimera 264 (89 min) than sANTXR2 (77 min) and the difference was highlysignificant (p=0.0046) suggesting increased therapeutic potency of themultivalent particles over monomeric sANTXR2 as an inhibitor of theanthrax toxin. It will be interesting to determine whether differentpharmacokinetic profiles are observed in vivo for sANTXR2 and chimera264.

TABLE 1 IN VIVO INTOXICATION EXPERIMENTS IN FISHER 344 RATS AverageMolar ratio of Survivors/ TTD ± SD Groups VWA_(ANTXR2):PA total (min)PBS NA 3/3 NA LeTx NA 0/5 69.2 ± 1.5  FHV wt + LeTx NA 0/3 67.7 ± 5.1 sANTXR2 + LeTx 2:1 5/5 NA sANTXR2 + LeTx 0.2:1   0/5  77 ± 4.6FHV-VWA_(ANTXR2) 264 + 2:1 5/5 NA LeTx FHV-VWA_(ANTXR2) 264 + 0.2:1  0/5 89.4 ± 5.4  LeTx TTD: Time to death. NA: not applicable. LeTx:Lethal toxin.PBS vehicle of injection as negative control, lethal toxin (20 μg (240pmol) of protective antigen (PA) and 8 μg (89 pmol) of lethal factor(LF), FHV wt was injected at a concentration of 21 μg/rat (480 pmol).Computational Models of PA Bound to FHV-VWA_(ANTXR2) Particles.

Results from the cell intoxication assays and LeTx challenge experimentsin rats suggested that the chimeric particles could bind PA, which inturn indicated that the inserted VWA domains were correctly folded inthe context of the FHV particle. The differential behavior ofFHV-VWA_(ANTXR2) chimeras 206 and 264 in cell intoxication assays,however, potentially reflected differences in the accessibility of PA tothe VWA domains on the surface of the particles. To further investigatethis, PA63, the proteolytically activated form of PA, wascomputationally docked onto the VWA domains of the pseudoatomic modelsof chimeras 264 and 206 using the X-ray structure of PA63 complexed withthe ANTXR2 VWA domain as a guide^(9,17). It was evident that chimera 264could accommodate significantly more PA molecules than chimera 206 giventhe wider spacing of the VWA domains on this particle (compare FIGS. 3 cand d). Specifically, each subunit at the fivefold axes and three of thesix subunits around the quasi-sixfold axes could bind PA63 withoutsteric interference, giving a total occupancy of 120 PA molecules perparticle. In contrast, due to the close juxtaposition of the VWA domainson chimera 206, a maximum occupancy of 60 PA molecules per particle waspredicted. These results were consistent with the observation that atwofold higher concentration of chimera 206 was required to protectcells from intoxication with PA/LF_(N)-DTA (FIG. 4). Assuming anoccupancy of up to 120 of the 180 VWA domains on chimera 264, theparticles were, however, more potent on a molar basis than sANTXR2. Thisenhancement could have been the result of polyvalent effects which areknown to enhance the biological activity of peptides and proteinsrelative to their monomeric state²¹.

FHV-VWA_(ANTXR2) Particles Serve as a Highly Effective Vaccine Platform.

The observation that FHV-VWA_(ANTXR2) chimera 264 functioned as abinding surface for multiple copies of PA suggested that a complex ofthe two components might constitute an effective antigen for inductionof PA-specific antibodies. To test this, complexes were prepared bymixing chimera 264 with an excess of PA83 and unbound PA83 was removedby ultracentrifugation. Electron microscopic analysis showed that PA83formed thin protrusions emanating from the capsid surface.

For immunogenicity studies, rats (4/group) received two s.c. injections(0 and 3 weeks) of either 2.5 μg PA83, 5.4 μg of particle-PA complex(molar equivalent of 2.5 μg PA83 assuming an occupancy of 120 VWAdomains), or 2.9 μg of chimera 264. No adjuvants were employed in theseexperiments. ELISA assay of pre- and post-inoculation sera showed thatanimals immunized with the complex had significantly higher levels ofanti-PA antibody than animals receiving PA alone both at week 3(p=0.0028 compared to PA alone) and after boosting (week 7; p=0.0118compared to PA alone) (FIG. 4A). No significant antibody responseagainst ANTXR2 VWA was detected in these animals, but a response againstFHV protein was observed after the boost in rats that were immunizedwith the complex (FIGS. 4B and C). Why animals immunized with chimera264 alone did not mount a similar immune response against FHV protein isnot clear. The presence of PA may somehow enhance the response to FHV ormay influence the localization or interaction of FHV particles withantigen-presenting cells causing a difference in the observed anti-FHVantibody titer.

To test whether the anti-PA antibodies were protective against anthraxtoxin, the rats were challenged by intravenous inoculation with 10 MLDsof LeTx. All animals that had been immunized with the particle-PAcomplex survived, whereas all but one of the animals in the PA83 groupdied (FIG. 4D). Survival versus death correlated well with the level ofanti-PA antibody detected in the sera. However, the animal in thePA-only group that survived LeTx challenge had a serologic response toPA that was well below that of a non-surviving animal in the same group(FIG. 4D).

Based on the observation that animals immunized with the particle-PAcomplex showed a significant level of anti-PA antibody as early as 3weeks after the first immunization (FIG. 4A), we investigated whetheranimals could be protected against LeTx after a single injection. Rats(5/group) were immunized once with increased doses of the particle-PAcomplex (10.8 μg), PA83 (5 μg), or chimera 264 (5.8 μg) as a control.After 3 weeks the animals were bled and challenged one week later with10 MLDs of LeTx. All rats that were immunized with the particle-PAcomplex survived, whereas all other animals died (FIG. 4F). Of thosethat died, one animal had a serologic antibody response to PA that wasgreater than that of two animals in the group of survivors (FIGS. 4E and4F) indicating that the magnitude of the antibody response is not areliable predictor of protection. Serial dilution assay showed that theaverage reciprocal anti-PA titer in animals immunized with theparticle-PA complex was 3240, and in vitro LeTx neutralization assaysconfirmed the neutralizing activity of these antibodies (ED50:54). Takentogether our results show that multivalent display of PA on theFHV-VWA_(ANTXR2) scaffold yields a significant advantage overmonovalent, soluble PA as an immunogen for anthrax toxin.

In this example we have provided and developed a novel reagent thatcombines the functions of anthrax antitoxin and vaccine in a singlecompound. It is based on multivalent display of the ANTXR2 VWA domain onthe surface of the icosahedral, insect nodavirus FHV. We demonstratedthat the recombinant VLPs protect cultured cells and rats from anthraxintoxication as efficiently as the highly potent sANTXR2 receptor decoyand that they induced a potent immune response against LeTx when coatedwith PA. This immune response was neutralizing in vitro and protectedanimals against LeTx challenge following a single administration withoutadjuvant.

The immunogenicity studies showed that polyvalent display of PA inducesa more potent immune response than monomeric, recombinant PA (rPA),which is currently being developed as a second-generation anthraxvaccine [15,23]. Ordered arrays of antigens permit particularlyefficient cross-linking of B cell receptors which in turn leads tofaster and more robust B cell proliferation [24,25,26]. Given theexceptionally tight binding of PA to ANTXR2 under natural conditions(K_(d)=170 pM) [21] it is reasonable that complexes formed betweenchimera 264 and PA are sufficiently stable to serve as an immunogen invivo. Results from in vitro cell intoxication experiments support this,showing that the complexes were stable for at least 40 hrs at 37° C.Naturally occurring PA neutralizing antibodies do not bind to thereceptor-binding surface of PA [27]. PA immobilized on these particlesshould be able to elicit a protective immune response. Indeed, ratssurvived lethal toxin challenge four weeks after a single injection ofthe VLP-PA complex, whereas animals injected with an equivalent amountof rPA died. This result suggested rapid production of neutralizingantibodies in the absence of adjuvant, two key goals for the developmentof third generation anthrax vaccines. No significant antibody responseto ANTXR2 was observed, presumably because there are only 2 amino aciddifferences between human ANTXR2 displayed on the particle andendogenous rat ANTXR2 [11].

It will be useful to characterize the neutralizing antibody response inindividual animals after primary and secondary immunization. It willalso be of interest to determine the mechanism by which toxinneutralization occurs. For example, there is a slight difference inantibody response after primary and secondary immunization and a widerange of antibody titers between individual animals (FIG. 4). It will beof interest to establish whether these differences correlate withepitope specificity or are based on other immunologic parameters. Inaddition, it is interesting to compare the findings in this example tothose obtained with a B. anthracis spore-challenge model.

Because the chimeric particles are expressed from an mRNA that containsonly the coding sequence of the modified FHV coat protein while allother FHV sequences are missing, the resulting virus-like particles arenot infectious and thus cannot replicate in mammalian tissues [19]. Evennative FHV particles are unable to initiate infection in mammals as theydo not carry the FHV receptor and because FHV cannot replicate attemperatures above 31° C. [28]. We have also demonstrated previouslythat FHV VLPs expressed from baculovirus vectors in Sf21 cells do notcontain baculoviral or cellular DNA [19], thus ruling out potentialintegration of foreign DNA into mammalian genomes. Based on theseproperties, the chimeric particles can be expected to have a desirablesafety profile for applications in animals and humans.

While the potency of the nanoparticles herein as a vaccine is mostlikely due to polyvalent display of PA, polyvalency is less of a factorin the function of the particles as an antitoxin, given the extremelyhigh affinity between PA and ANTXR2. Moreover, since PA binds as amonomer to the particles, little, if any polyvalent effect is to beexpected. In fact, we detected no significant difference in IC₅₀ whencomparing nanoparticles with soluble ANTXR2 in cell intoxication assays.However, the use of polyvalency to increase the affinity between aligand and its target receptor is a useful general strategy [31].Recently, Rai et al. [32] reported that “pattern matching” is a usefulparameter for polyvalency to reach its maximum potential.

In vivo potency of viral nanoparticles is also determined in part bytheir pharmacokinetic parameters. Such parameters have recently beenreported for viral nanoparticles derived from the plant virus cowpeamosaic virus [33]. It will be useful to determine any differences inplasma clearance kinetics and biodistribution of soluble ANTXR2 versusANTXR2-containing nanoparticles.

The VWA domain of ANTXR2 was a candidate for insertion into a loop ofthe FHV coat protein because the N and C termini are only separated by4.8 Å in the native structure [34]. In addition, this domain adopts acompact Rossmann-like α/β-fold that can evidently form independentlywithin the context of a larger protein while not interfering withaccurate folding of the carrier protein. This hypothesis was supportedby the observation that the high-resolution structure of the VWA domaincould be fitted easily into the cryoEM density maps. To our knowledge,Hepatitis B virus is the only other virus for which icosahedral surfacedisplay of an entire protein in its biologically active conformation hasbeen demonstrated. In that case, genetic insertion of the greenfluorescent protein in a surface-exposed loop of the core proteinresulted in efficient formation of fluorescent HBV capsids [35].

In principle it should be possible to expand the use of the FHV platformto display additional anthrax antigens either in the presence or absenceof the ANTXR2 VWA domain. Specifically, direct insertion of peptides orentire domains derived from PA, LF and EF is feasible as long as thetermini of the domains are in close enough proximity for insertion intothe FHV coat protein loops. It is also possible that the two insertionsites at positions 206 and 264 could be used in combination to createparticles with multiple functionalities. This could greatly enhance theprotection afforded by the resulting particles.

Numerous other strategies are being pursued to develop improved anthraxvaccines, including PA-expressing Salmonella [36] and B. subtilis [37],adenovirus encoding PA domain 4 [38], rabies virus encoding GP-PA fusionprotein [39] and bacteriophage T4 particles decorated with PA-hoc fusionproteins [40,41,42]. None of these, however, combine the function ofvaccine and antitoxin as in the present invention. In those cases whereimmunized animals were challenged with LeTx or anthrax spores, only theadenovirus construct provided complete protection after a singleimmunization [38]. One strategy involves non-covalent surface display ofintact proteins and protein complexes on bacteriophage T4 particles. Theprolate lattice of the T4 capsid permits efficient surface presentationof anthrax toxin through in vitro addition of Hoc- and/or Soc proteinfusions with PA, LF, or EF to hoc⁻soc⁻ phage either separately or incombination [40,42]. Mice immunized with phage displaying PA, EF and LFgenerated high levels of neutralizing antibodies [41] but results fromtoxin or spore challenge experiments have not yet been reported.

In summary, we have developed a reagent that serves a dual purpose incombating B. anthracis infection. It functions as a competitiveinhibitor of anthrax toxin in vivo suggesting that it could be useful asa therapeutic compound, particularly in combination with standardantibiotic therapy. In addition, when complexed with PA, it hassignificant advantages as an immunogen compared to monomeric PA and thusforms the basis for development of an improved anthrax vaccine.

Further Example Details

Construction of recombinant baculoviruses. DNA fragments encoding FHVcoat protein-ANTXR2 VWA domain chimeras were generated by overlapextension PCR using Pfu polymerase [43]. Three DNA fragments containingthe nucleotide sequence for the N-terminal portion of the coat protein,the ANTXR2 VWA domain (GenBank accession no. AY23345, nts 115-657, aminoacids 38-218) and the C-terminal portion of the coat protein, wereinitially generated. The template used for generating segmentscontaining the FHV coat protein sequence was plasmid pBacPAK9RNA2□[44],which contains the full-length cDNA of FHV RNA2 in baculovirus transfervector pBacPAK9 (BD Biosciences). The template used for generating thesegment containing the ANTXR2 VWA coding sequence was a derivative ofplasmid PEGFP-N1 [8]. Following overlap extension PCR, the full-lengthproduct was digested with BamH1 and XbaI, gel-purified and ligated intoequally digested pBacPAK9. The transfer vector was amplified in E. colistrain DH5α, purified and sequenced to confirm the presence of theANTXR2 VWA sequence and to ensure the absence of inadvertent mutations.Recombinant baculoviruses AcFHV-VWA_(ANTXR2)-264 andAcFHV-VWA_(ANTXR2)-206 were generated by transfecting Sf21 cells with amixture of transfer vector and linearized Bsu36I-linearized BacPAK6 (BDBiosciences) baculovirus DNA as described previously [43].

Cells and infection. Spodoptera frugiperda cells (line IPLB-Sf21) werepropagated and infected as described previously [19]. Trichoplusia nicells were propagated and infected as described by Dong et al. [45]except that EX-CELL 401 medium was replaced with ESF921 (ExpressionSystems).

Purification of virus-like particles (VLPs). VLPs were purified from T.ni suspension cultures 5 to 6 days after infection. NP-40 substitute(Fluka) was added to the culture to a final concentration of 1% (v/v)followed by incubation on ice for 10-15 min. Cell debris was pelleted bycentrifugation in a Beckman JA-14 rotor at 15,300×g for 10 minutes at 4°C. VLPs in the supernatant were precipitated by addition of NaCl to afinal concentration of 0.2 M and polyethylene glycol 8000 (Fluka) to afinal concentration of 8% (wt/vol) and stirring the mixture at 4° C. forone hour. The precipitate was collected by centrifugation at 9632×g for10 min at 4° C. in a JA-14 rotor and resuspended in 50 mM HEPES buffer,pH 7.5. Insoluble material was removed by centrifugation at 15,300×g for20 min at 4° C. VLPs in the clarified supernatant were pelleted througha 4-ml 30% (wt/wt) sucrose cushion in 50 mM Hepes, pH 7.5. bycentrifugation in a Beckman 50.2 Ti rotor at 184,048×g for 2.5 h at 11°C. The pellet was resuspended in 50 mM Hepes buffer, pH 7.5 and loadedonto a 10-40% (wt/wt) sucrose gradient in the same buffer. The gradientswere spun in a Beckman SW 28 rotor for 3 h at 103,745×g. VLPs werecollected from the gradient by inserting a needle below the VLP band andaspirating the material into a syringe. Alternatively, gradients werefractionated with continuous absorbance at 254 nm on an ISCO gradientfractionator at 0.75 ml/min and 0.5 min per fraction. Fractionscontaining VLPs were then dialyzed against 50 mM Hepes, pH 7.5 andconcentrated to 1-5 mg/ml using a centrifugal concentrator with a100,000 MW cut off (Amicon, Millipore). The final protein concentrationwas determined by BCA assay (Pierce Chemicals) and purity was evaluatedby densitometry (Fluor Che™SP, Alpha Innotech) after electrophoresis ona 10% Bis-Tris gel stained with Simply Blue (Invitrogen).

Electron microscopy. Samples of gradient-purified VLPs were negativelystained with 1% (wt/v) uranyl acetate. A drop of each sample wasadsorbed to a glow-discharged, collodion-covered copper grid and allowedto adsorb for 1-2 min. Excess solution was removed by blotting withfilter paper. The grids were washed and blotted with filter paper threetimes by floating on droplets of 50 mM Hepes, pH 7.5. Each grid was thentreated three times with a drop of 1% uranyl acetate solution and leftin the third drop for 1-2 min prior to blotting and air drying. Thesamples were viewed in a Philips/FEI CM 100 transmission electronmicroscope at 100 kV.

Electron cryomicroscopy and image reconstruction. Frozen-hydratedsamples were prepared using standard methods [46]. In brief, an aliquotof the sample was applied to a glow-discharged Quantifoil holeycarbon-coated grid (2/4 Cu—Rh), blotted with filter paper, and rapidlyplunged into liquid ethane. Low-dose electron micrographs ofFHV-VWA_(ANTXR2)-264 VLPs were recorded onto Kodak SO163 film at amagnification of 45,000× on a Philips/FEI CM120 transmission electronmicroscope. For FHV-VWA_(ANTXR2)-206 VLPs, low-dose micrographs wererecorded on a CCD camera at a magnification of 50,000× on a Philips/FEITecnai20 transmission electron microscope. The grids were maintained at−180° C. using a Gatan 626 cryo-stage. Micrographs with minimalastigmatism and drift, as assessed by visual inspection and opticaldiffraction, were digitized with a Zeiss microdensitometer (Z/I Imaging)giving a step-size of 3.1 Å on the specimen. Images recorded on the CCDcamera had a step-size of 2.26 Å. Particle images were extracted withthe program X3D[47] and were processed by polar Fourier transformmethods using the program PFT[48]. A previously calculated model of wtFHV [49] was used as the starting model. Initial refinement cycles wererestricted to the radii spanning the FHV capsid and then relaxed toincorporate the extra domains. Using a Fourier shell correlation cut-offvalue of 0.5, the FHV-VWA_(ANTXR2)-206 and FHV-VWA_(ANTXR2)-264 mapswere refined to resolutions of 25 and 23 Å, respectively.

Generation of pseudoatomic models. The atomic coordinates of the FHVcoat protein subunit and the VWA domain of ANTXR2 (PDB ID: 1SHT) wereused to generate a pseudoatomic model of the FHV-VWA_(ANTXR2)-206/264virus-like particles. Specifically, the models were created with theprogram O [50] by visually positioning the ANTXR2 VWA domains at thesurface of the FHV structure and adjusting for overlap. The models werethen further refined against the structure factor amplitudes derivedfrom the cryo-EM density using the program CNS [51]. Individual subunitsand domains of the FHV-VWA_(ANTXR2) chimera were allowed to moveindependently as rigid bodies and subjected to five rounds of 20 cyclesof rigid body refinement. Protective antigen 83 (PA83) molecules weredocked onto the resulting FHV-VWA_(ANTXR2)-206/264 models using thestructure of PA63 complexed with the ANTXR2 VWA domain (PDB-ID: 1T6B) asa guide [9]. Once all 180 ANTXR2 VWA domains on the FHV-VWA_(ANTXR2)chimera were populated with PA83 molecules, a minimal number of PAmolecules were selectively removed to relieve steric clashes withneighboring PA molecules.

Quantification of PA83 bound to chimeras 206 and 264. Recombinant PA83(List Biological Laboratories, Inc, CA) in 5 mM Hepes, 50 mM NaCl, pH7.5 was mixed with purified chimeras 206 and 264 in 50 mM Hepes, pH 7.5in a ratio of 180:1 (equimolar amounts of PA83 and VWA domains).Following incubation for 20 min. at room temperature an aliquot fromeach of the samples was removed and stored at −20° C. pending analysis.The remainder of the samples was transferred to an ultracentrifuge tubeand underlayed with a 30% (wt/wt) sucrose cushion in 50 mM Hepes pH 7.5.Complexes of chimeras decorated with PA83 were pelleted bycentrifugation at ˜200,000×g for 45 min. The complexes were resuspendedin 50 mM Hepes, mixed with SDS loading buffer and heated at 95° C. for10 minutes. Aliquots were electrophoresed through a 4-12% Bis-Trispolyacrylamide gel, in parallel with the aliquots taken beforepelleting. The gels were stained with Simply Blue (Invitrogen). Theamount of protein in each bands was determined by densitometric analysisusing Fluor Chem SP (Alpha Innotech).

Cell intoxication assay with CHO-K1 cells. Cell intoxication studieswere performed in CHO-K1 cells as described previously [10]. Briefly,5×10³ cells in 100 μl Hams-F12 nutrient mixture (Gibco BRL) supplementedwith 10% fetal bovine serum were plated into wells of a 96-wellmicrotitre plate a day prior to the assay. Varying amounts ofFHV-VWA_(ANTXR2) VLP or soluble ANTXR2 [8] were preincubated for 20 min.in 100 μl medium containing PA and LF_(N)-DTA at a molar concentrationof 10⁻⁸ and 10⁻¹⁰, respectively. The mixture was added to the cells,which were incubated at 37° C. for approximately 40 hrs. The medium wasthen replaced with 50 μl Celltiter-glo reagent (Promega) diluted 1:1with PBS. Luciferase activity as a measure of cell viability wasdetermined with a luminometer (TopCount NXT, Perkin Elmer). Non-linearregression analysis was used to calculate IC₅₀ values (PRISM, GraphPad).

Macrophage-based toxin neutralization assay. RAW264.7 cells (5×10⁴) wereplated in each well of a white 96-well tissue culture plate (CorningCostar) with 100 μl Dulbecco's Modified Eagle Medium (Gibco)supplemented with 10% standard fetal bovine serum the day before theassay. Varying amounts of FHV-VWA_(ANTXR2) VLP were preincubated for 20min. in 400 μl medium containing PA and LF at a molar concentration of10⁻⁸ and 10⁻⁹, respectively. The mixture (100 μl) was added to the cellsin triplicate and incubated at 37° C. for approximately 5 hrs. Cellviability was determined as described for CHO-K1 cells.

Toxin neutralization assay. RAW264.7 cells (10⁴) were plated in eachwell of a white 96-well tissue culture plate (Corning Costar) with 100μl Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10%standard fetal bovine serum the day before the assay. Individual serafrom animals vaccinated either with PA83 or FHV-VWA_(ANTXR2)-PA83complex were pooled and serial dilutions made in DMEM were mixed with100 μl DMEM containing PA and LF at a molar concentration of 10⁻⁸ and10⁻⁹, respectively. The mixture was pre-incubated for 1 hr at roomtemperature and then added to the cells. Each sample was tested intriplicate. Cells were incubated at 37° C. for approximately 24 hrs. Themedium was then replaced with 50 μl Celltiter-glo reagent (Promega)diluted 1:1 with PBS. Luciferase activity as a measure of cell viabilitywas determined with a luminometer (TopCount NXT, Perkin Elmer). Toxinneutralization titer was reported as the reciprocal dilution at which50% of the cells were protected from LeTx.

LeTx challenge with FHV-VWA_(ANTRX2) VLPs. LeTx challenge experimentswere performed in cannulated, male Fisher 344 rats (180-200 g, Harlan,Indianapolis, Ind.) according to protocols approved by the ScrippsInstitutional Animal Care and Use Committee. LeTx for each rat wasprepared by mixing 20 μg PA and 8 μg LF (List Biological Laboratories,Campbell, Calif.). All rats were anesthetized with isofluorane andinoculated through a jugular vein cannula with 500 μl of LeTx (control),or LeTx premixed with FHV-VWA_(ANTXR2) VLPs or sANTXR2 (test).Additional rat control groups were injected with either PBS or wild-typeFHV. Rats recovered from anesthesia within five minutes after dosing andwere monitored for symptoms of intoxication (agitation, respiratorydistress, hypoxia) and death as determined by cessation of respiration.

Statistical analysis. Data are presented as mean ±S.D. Significance wasreported using the Student's unpaired T-test (Prism, GraphPad SoftwareInc., San Diego Calif.). p values<0.05 were considered statisticallysignificant. Data were also analyzed using 2-way ANOVA followed byTukey's post-comparison test to confirm significance.

Preparation of FHV-VWA_(ANTRX2) chimera 264-PA83 complexes. Purifiedchimera 264 VLPs in 50 mM Hepes, pH 7.5 were mixed with a 4-fold molarexcess of recombinant PA83 (List Biological Laboratories, Inc, CA) in 5mM Hepes, 50 mM NaCl, pH 7.5 and incubated at room temperature for 20min with mild agitation. The sample was then transferred to anultracentrifuge tube and underlayed with a 30% (wt/wt) sucrose cushionin 50 mM Hepes pH 7.5. Complexes of chimera 264 decorated with PA83 werepelleted by centrifugation at 197,568×g at 11° C. in an SW41Ti rotor for1.5 h or an SW 55 Ti rotor for 45 min. The complexes were resuspended in50 mM Hepes and analyzed by electrophoresis to confirm the presence ofchimera 264 and PA83 proteins.

Rat Immunization and LeTx challenge. Immunization studies were performedin male Harlan Sprague Dawley (HSD) rats (180-200 g, Harlan,Indianapolis, Ind.) according to protocols approved by the ScrippsInstitutional Animal Care and Use Committee. For double doseimmunization, rats were injected s.c. with 200 μl containing either 2.5μg of PA83 (List Biological Laboratories, Inc, CA), 5.4 μg ofFHV-VWA_(ANTXR2)-264-PA83 (molar equivalent of 2.5 μg PA83), or 2.9 μgFHV-VWA_(ANTXR2)-264 (molar equivalent of particles complexed with PA83)all prepared in PBS. For single dose immunization, rats were injecteds.c. with 200 μl containing either 5 μg of PA83, 10.8 μg ofFHV-VWA_(ANTXR2)-264-PA83 (molar equivalent of 5 μg PA83), or 5.8 μg ofFHV-VWA_(ANTXR2)-264 (molar equivalent of particles complexed with PA83)all in PBS. Rats were anesthetized with isofluorane before allprocedures. Serum was prepared from blood (200 μl per rat) collectedfrom the retro-orbital plexus before immunization, three weeks postprimary immunization, and four weeks post secondary immunization (study1 only). LeTx challenge experiments were performed 13 weeks post initialimmunization for the double dose immunization study and 4 weeks postimmunization for the single dose immunization study. LeTx for each ratwas prepared by mixing 40 μg PA and 8 μg LF (List BiologicalLaboratories, Campbell, Calif.) in PBS. Rats were anesthetized withisofluoranes and inoculated with 500 μl of LeTx or PBS (control) byintravenous tail vein injection. Rats recovered from anesthesia withinfive minutes after dosing and were monitored for symptoms ofintoxication and death as described above.

Antibody Detection. Serum samples were tested for antibody response toPA, ANTXR2, and FHV by ELISA assays. Briefly, microtitre Immulon 2HB96-well plates (Dynex Technologies, Inc.) were coated with 100 μl of 10μg/ml PA83, sANTXR2, or FHV in coating buffer (0.1M NaHC03 pH 8.5),blocked with 3% non-fat milk in TBS, and incubated with serum samplesdiluted 1:100 and 1:1,000 in 1% non-fat milk in TBS pH 7.0 with 0.05%Tween 20 for 1 hour at room temperature. After washing, wells wereincubated with biotin-SP-conjugated donkey anti-rat IgG (JacksonImmunoresearch, West Grove, PA) diluted 1:20,000 for 1 hour at roomtemperature. Plates were washed and incubated for 45 minutes withstreptavidin-alkaline phosphatase conjugate (GE Healthcare Bio-SciencesCorp., Piscataway, NJ) diluted 1:5,000 in TBS. After washing, plateswere incubated with p-nitriphenyl phosphate (Sigma-Aldrich, St. Louis,MO) at 37° C. for 20 minutes. 2N NaOH was added to stop the reaction andthe signal was quantified using an ELISA reader (Molecular Devices,Sunnyvale, Calif.) at 405 nm.

AMINO ACID SEQUENCE OF FHV-CMG2 CHIMERA 264(SEQ ID NO 1).CMG2 SEQUENCE IS UNDERLINED.MVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNRRRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRKDVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTSMFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQFPVATDPATSSLVHTLVGLDGVLAVGPDNGSESFIKGVFSQSACNEPDFEFNDILEGIQTLPPSCRRAFDLYFVLDKSGSVANNWIEIYNFVQQLAERFVSPEMRLSFIVFSSQATIILPLTGDRGKISKGLEDLKRVSPVGETYIHEGLKLANEQIQKAGGLKTSSIIIALTDGKLDGLVPSYAEKEAKISRSLGASVYCVGVLDFEQAQLERIADSKEQVFPVKGGFQALKGIINSILAQSCSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIRVSAPEGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVARSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGFGFAMINO ACID SEQUENCE OF FHV-CMG2 CHIMERA 206(SEQ ID NO: 2). CMG2 SEQUENCE IS UNDERLINEDMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNRRRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRKDVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTSMFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQFPVATSCRRAFDLYFVLDKSGSVANNWIEIYNFVQQLAERFVSPEMRLSFIVFSSQATIILPLTGDRGKISKGLEDLKRVSPVGETYIHEGLKLANEQIQKAGGLKTSSIIIALTDGKLDGLVPSYAEKEAKISRSLGASVYCVGVLDFEQAQLERIADSKEQVFPVKGGFQALKGIINSILAQSCAEATSSLVHTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEPDFEFNDILEGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIRVSAPEGAVNSAILKAWSCIEYRPNPNALMLYQFGHDSPPLDEVALQEYRTVARSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGFGFCDNA SEOUENCE OF FHV RNA2 (SEQ ID NO: 3)GTAAACAATTCCAAGTTCCAAAATGGTTAATAACAACAGACCAAGACGTCAACGAGCTCAACGCGTTGTCGTCACAACAACCCAAACAGCGCCTGTTCCACAGCAAAACGTGCCACGTAATGGTAGACGCCGACGTAATCGCACGAGGCGTAATCGCCGACGTGTGCGCGGAATGAACATGGCGGCGCTAACCAGATTAAGTCAACCTGGTTTGGCGTTTCTCAAATGTGCATTTGCACCACCTGACTTCAACACCGACCCCGGTAAGGGAATACCTGATAGATTTGAAGGCAAAGTGGTCAGCCGAAAGGATGTCCTCAATCAATCTATCAGCTTTACTGCCGGACAGGACACTTTTATACTCATCGCACCTACCCCCGGAGTCGCCTACTGGAGTGCTAGCGTTCCTGCTGGTACTTTTCCTACTAGTGCGACTACGTTTAACCCCGTTAATTATCCGGGTTTTACATCGATGTTCGGAACAACTTCAACATCTAGGTCCGATCAGGTGTCCTCATTCAGGTACGCTTCCATGAACGTGGGTATTTACCCAACGTCGAACTTGATGCAGTTTGCCGGAAGCATAACTGTTTGGAAATGCCCTGTAAAGCTGAGTACTGTGCAATTCCCGGTTGCAACAGATCCAGCCACCAGTTCGCTAGTTCATACTCTTGTTGGTTTAGATGGTGTTCTAGCGGTGGGGCCTGACAACTTCTCTGAGTCATTCATCAAAGGAGTGTTTTCACAGTCGGCTTGTAACGAGCCTGACTTTGAATTCAATGACATATTGGAGGGTATCCAGACATTGCCACCTGCTAATGTGTCCCTTGGTTCTACGGGTCAACCTTTTACCATGGACTCAGGAGCAGAAGCCACCAGTGGAGTAGTCGGATGGGGCAATATGGACACGATTGTCATCCGTGTCTCGGCCCCTGAGGGCGCAGTTAACTCTGCCATACTCAAGGCATGGTCCTGCATTGAGTATCGACCAAATCCAAACGCCATGTTATACCAATTCGGCCATGATTCGCCTCCTCTCGATGAGGTCGCGCTTCAGGAATACCGTACGGTTGCCAGATCTTTGCCGGTTGCAGTGATAGCGGCCCAAAATGCATCAATGTGGGAGAGAGTGAAATCCATCATTAAATCCTCCCTGGCTGCTGCAAGCAACATTCCCGGCCCGATCGGTGTCGCCGCAAGTGGTATTAGTGGACTGTCAGCCCTTTTTGAAGGATTTGGCTTTTAGAAGCATCCGGACGCCAACCTAACCGGGCAAGTATCCGAACAATCGGACATTTGGCCACAATAAGCCCAATTTGGTTGAAGATTAAAGTAGTGAGCCCCCTTAGCGCGAAACCGGAATTTATATTCCAAACCAGTTTAAGTCAACAGACTAAGGT

ACCESSION NUMBERS

1. Genbank Please see the genbank website on the worldwide web atncbi.nlm.nih.gov/genbank.

Human capillary morphogenesis protein 2 (CMG2): AY23345

Flock House virus coat protein: NC004144.

2. Protein Data Bank See the worldwide web at rcsb.org/pdb.

VWA domain of CMG2: 1SHT

B. anthracis protective antigen PA63 complexed with CMG2: 1T6B

3. Virus Particle Explorer See the viperdb.scripps.edu. website on theworldwide web.

Flock House virus particle coordinates

Figure Legends for Example 1

FIG. 1. (a) Ribbon diagram of a single FHV coat protein subunit showingthe surface-exposed loops into which the VWA domain of ANTXR2 wasinserted. (b) Schematic diagram showing structure of FHV-VWA_(ANTXR2)chimeric proteins 206 (top) and 264 (bottom). Numbers refer to aminoacid positions in the FHV coat protein. The assembly-dependentautocatalytic maturation cleavage occurs between Asn 363 and Ala 364.(c) Electrophoretic analysis of purified VLPs on a 10% Bis-Tris gelstained with Simply Blue (Invitrogen). Lane 1, molecular weight markers;lane 2, wt FHV VLPs; lane 3, FHV-VWA_(ANTXR2) chimera 206; lane 4,FHV-VWA_(ANTXR2) chimera 264. (d) Electron micrographs ofgradient-purified wt and chimeric VLPs negatively stained with uranylacetate. Bar=500 Å.

FIG. 2. Dose response curve showing cell viability as a function ofantitoxin concentration. CHO-K1 cells were incubated with PA-LF_(N)DTAin the presence of increasing concentrations of sANTXR2, chimera 206, orchimera 264. After incubation at 37° C. for 48 hr, cell viability wasdetermined with Celltiter-glo (Promega). Data points represent the mean±SD values for triplicate samples. All data were normalized to untreatedcell controls.

FIG. 3. (a and b) Pseudoatomic models of FHV-VWA_(ANTXR2) chimeras.X-ray coordinates of FHV capsid protein (green) and ANTXR2 VWA domain(yellow) were docked into the cryo-EM density of chimera 206 (a) andchimera 264 (b). Panels show surface views of the particles in theabsence of the cryoEM density maps. Note the different distributions ofthe ANTXR2 domains on the surfaces of the VLPs. (c and d) In silicomodel of PA83 bound to the surface of FHV-VWA_(ANTXR2) chimeras. PA83(purple) was modeled onto the surface of chimera 206 (c) and chimera 264(d) using the known high resolution X-ray structure of theANTXR2-VWA/PA83 complex as a guide [34]. Panels show surface views ofthe entire particles to illustrate the difference in occupancy of PA83on the two chimeras.

FIG. 4. Antibody and lethal toxin challenge response of immunized rats.(a-d) Rats (four per group) were immunized with FHV-VWA_(ANTXR2)-PA83complex, FHV-VWA_(ANTXR2) chimera alone, PA83 or PBS and boosted fourweeks later. Serum samples were collected prior to as well as 3 and 7weeks after immunization and tested by ELISA for IgG-specific antibodyresponse to (a) PA, (b) VWA_(ANTXR2) and (c) FHV coat protein. Datarepresent the mean ±SD of animals in the respective groups and are shownfor the 1:1000 serum dilution in panels (a) and (b) and for the 1:100serum dilution in panel (c). In panel (a) at week 3, (*) indicatesp=0.003 compared to PBS control and (**) indicates p=0.003 compared toPA83 alone. At week 7, (*) indicates p=0.005 compared to PBS control and(**) indicates p=0.012 compared to PA83 alone. (d) Relationship betweenanti-PA antibody level and survival of individual rats followingchallenge with 10 MLDs of LeTx. (e-f) Rats (five per group) wereimmunized once with FHV-VWA_(ANTXR2)-PA83 complex, FHV-VWA_(ANTXR2)chimera alone, PA83 or PBS. Serum samples were collected prior to and 3weeks after immunization, diluted 1:100, and tested for IgG-specificantibody response to PA (e). Data represent the mean ±SD of animals inthe respective groups. At week 3, (*) indicates p<0.0001 compared to PBScontrol and (**) indicates p=0.001 compared to PA83 alone. (f)Relationship between anti-PA antibody level and survival of individualrats following challenge with 10 MLDs of LeTx.

FIG. 5. 3D surface-shaded reconstructions of (a) FHV-VWA_(ANTXR2)chimera 206, (b) FHV-VWA_(ANTXR2) chimera 264 and (c) wt FHV. Note theprotruding surface densities on the chimeric VLPs due to the addition ofthe VWA domain of ANTXR2. Bar=100 Å.

FIG. 6. Pseudoatomic models of FHV-VWA_(ANTXR2) chimeras. X-raycoordinates of FHV capsid protein (green) and ANTXR2 VWA domain (yellow)were docked into the cryo-EM density (grey) of chimera 206 (a) andchimera 264 (b). Shown are octants of the density maps to illustrate thegood fit of the high resolution X-ray structures into the cryoEM maps.Density within the interior of the capsid shell is ascribed to theencapsidated RNA.

FIG. 7. In silico model of PA83 bound to the surface of FHV-VWA_(ANTXR2)chimeras. A maximum number of PA83 molecules (purple) was modeled ontothe surface of chimera 206 (a) and chimera 264 (b), as limited by sterichindrance. Modeling was based on the known X-ray crystal structures ofFHV [17] and ANTXR2-VWA/PA63 complex [9]. The FHV capsid protein and theVWA domain of ANTXR2 are in green and yellow, respectively. Panels showcross-sections of the chimeric particles, including the cryoEM densitymap (grey).

FIG. 8. Quantification of PA83 bound to chimera 206 and chimera 264. (a)Recombinant PA83 was mixed with purified chimeras 206 and 264 in a ratioof 180:1 (equimolar amounts of PA83 and VWA) and an aliquot from eachsample was removed and stored. The remainder of the sample wascentrifuged through a sucrose cushion to remove unbound PA83 and thepellet was resuspended in electrophoresis buffer. Aliquots wereelectrophoresed through a 4-12% Bis-Tris polyacrylamide gel in parallelwith aliquots taken before pelleting. The gels were stained with SimplyBlue (Invitrogen). Lane 1, molecular weight markers; lane 2, PA83 andchimera 206 before pelleting; lane 3, PA83 and chimera 206 afterpelleting; lane 4, PA83 and chimera 264 before pelleting; lane 5, PA83and chimera 264 after pelleting. (b) Quantitative representation of PA83associated with chimeric particles after pelleting. Protein representingPA83 and chimeras 206 and 264 shown in panel (a) was quantified bydensitometric analysis using Fluor Chem SP (Alpha Innotech) and thenumber of PA molecules bound to the particles calculated. The numberswere normalized to the amount of protein in the respective samplesbefore pelleting.

For relevant example details see also: Manayani et al. (2007) A ViralNanoparticle with Dual Function as an Anthrax Antitoxin and Vaccine PLoSPathog. 2007 3 (10):e142 (doi:10.1371/journal.ppat.0030142),incorporated herein by reference in its entirety.

Numbered References for Example 1 and Above

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Example 2 Other Applications of the VNI-Based Multivalent Platform:Combination Vaccines to Simultaneously Provide Protection AgainstMultiple Pathogens or Toxins

An attractive feature of the VNI platform is that, through the use ofchimeric proteins with PA fused to other immunogens, it should bepossible to use this as a versatile technology to generate rapidlyacting and effective vaccines against other pathogens and theirproducts. These other pathogens include category A, B, and C agents,some of which represent major bioterrorism threats. For example, therehave been several recent advances in the development of mucosal vaccinesagainst ricin and botulinum neurotoxins. However, in each case effectiveimmunization involves multiple dosings with the immunogen (22-24).

As shown in FIG. 9, the current example includes using the VNI platformto present multiple immunogens to the immune system. The strategy, inthis example, is to fuse PA with antigenic portions of ricin orbotulinum toxins (e.g. PA-R and PA-B, respectively). These fusionproteins are then multivalently arrayed on the surface of VNIs togenerate combination vaccines.

The VNI-multivalent vaccine approach can circumvent the need formultiple dosings of immunogen, giving rise to a more rapid and efficientresponse. Moreover, by combining PA fusion proteins with the C-terminaldomains of multiple BoNT serotypes (e.g. A, B, and E), it should bepossible to use the VNI approach toward development of multivalent BoNTvaccines. This approach can be used to generate combination vaccines,which can simultaneously provide protection against, e.g., anthrax,botulinum A toxin (BoNT/A), and ricin toxin. In particular, by fusingimmunogenic portions of other toxins such as ricin toxin or botulinumtoxin with PA, the VNI platform is used to immunize against multipletoxins simultaneously (FIG. 9).

Use of the VNI platform to develop combination vaccines for additionalpathogens. Here the ability to generate a combination VNI-based vaccineis discussed. Fusion proteins of PA and ricin toxin or botulinum A toxinare generated and arrayed on the surface of VNIs (FIG. 10). Immunizationand challenge studies can be used to monitor immunity simultaneouslygenerated against the toxins.

Generating an oligomerization- and cleavage-defective PA mutant. In thisexample, the use of the multivalent VNI platform employs alteredversions of PA in which domain 1, also known as PA₂₀, is replaced withmoieties from botulinum neurotoxin A and ricin A chain. To ensure thatthese heterologous moieties remain stably associated with PA and are notproteolytically removed by furin, a mutant version of PA is used inwhich the furin cleavage site is replaced with the amino acids SNKE.This mutation was previously shown to inhibit furin cleavage of PA (32).In addition, to ensure that the PA fusion proteins do not spontaneouslyheptamerize (a process normally inhibited in-cis by domain 1), an Asp toLys mutation is introduced at position 512. This amino acid alterationinhibits PA heptamerization without compromising overall structure orreceptor binding of PA (33). The mutations are engineered into wt PAusing standard molecular cloning procedures. A cDNA of wt PA iscurrently available in the E. coli expression vector pET22b. The PAcontaining modifications at the furin cleavage site and at residue 512are used throughout the example.

Generating and purifying PA-Ricin (PA-R) fusion protein from E. coli.Standard molecular cloning procedures are employed to replace the codingsequence of domain 1 (amino acids 1-167) in PA with that of ricin toxinA chain (RTA), to generate the PA-R fusion protein. A His-tag is addedto the N terminus of RTA to facilitate purification of PA-R. The PA-Rfusion construct is expressed in E. coli using vector pET22b. Protocolsthat closely follow those currently used for expression of wt PA areemployed. The fusion protein is purified by affinity chromatographyusing a nickel column and, if necessary, by FPLC using ion exchange orgel filtration chromatography. Purified protein is aliquoted and storedat −80° C.

Generating and purifying PA-botulinum (PA-B) fusion protein from E.coli. The carboxyterminal portion of the heavy chain of botulinumneurotoxin A (BoNT(A)), amino acids 873-1295 (H_(C) region), are used toreplace domain I of PA, to generate the PA-B fusion protein. Inaddition, an N terminal His tag is added to the BoNT(A) moiety tofacilitate purification of the fusion protein. The PA-B fusion constructis expressed in E. coli using standard procedures that closely followthose currently used for expression of wt PA. The fusion protein ispurified by affinity chromatography using a nickel column and, ifnecessary, is further purified by FPLC using ion exchangechromatography. Purified PA-B is aliquoted and stored at −80° C.

Verifying correct folding of PA-R and PA-B. Correct folding of RTA inthe context of PA-R is verified by confirming the enzymatic activity ofthe protein. To this end, purified PA-R is added to a cell-free lysateand inhibition of protein synthesis will be measured. Specifically, PA-Rwill be added to a rabbit reticulocyte lysate in the presence of³⁵S-methionine and mRNA, and incubated for 2 h at 37° C. Purified RTAchain (Vector Laboratories) is used as a positive control. Negativecontrol samples do not receive toxin. Protein is precipitated withtrichloroacetic acid and the amount of radioactivity incorporated isdetermined by liquid scintillation counting. Correct folding of theBoNT(A) domain is confirmed by immunoprecipitation with aconformation-specific antibody. Specifically, monoclonal antibody CR1(34) is employed. This antibody was shown to bind with high affinity toa discontinuous epitope comprised of loops located in both the H_(CN)and H_(CC) domains. Incorrectly folded BoNT(A) H_(C) domain is highlyunlikely to bind to CR1. Alternatively, correct folding of BoNT(A) canbe verified by confirming its function as a competitive inhibitor in thebotulinum neurotoxin neutralization assay.

Scale-up and purification of PA-fusion proteins. The PA-R and PA-Bfusion proteins are scaled up and purified from the periplasm of E. coliBL21 cells as described previously (35). These proteins are purified tohomogeneity by FPLC with HiTrap QFF and Superose 12 (Amersham), orHiLoad Superdex 200 (Amersham), columns. The relative protein purity isthen established by ImageQuant analysis (Amersham) of Coomassie-stainedprotein samples after SDS-PAGE as described before (35)).

Decorate VNI with PA fusion proteins and determine bindingstoichiometry. VNI particles are decorated with PA-R and PA-B fusionproteins using the same protocol employed for decoration of VNI with wtPA. Integrity of decorated particles is verified by electron microscopyusing negatively stained samples. The number of PA-R and PA-B moleculesbound to VNI is determined biochemically using gel electrophoresis ofpurified complexes and quantification of bands corresponding to PA-R,PA-B and VNI as was done previously for VNI-PA.

Immunization of mice with VNI-PA-R and VNI-PA-B. Experiments employinglive animals are conducted with a derivative of PA-R that contains twoamino acid mutations in the ricin A chain (Y80A/V76M). Mutation Y80Aprevents local vascular leakage in vaccinated individuals and mutationV76M inactivates the enzymatic activity of RTA. This double mutant ofRTA was shown to be safe and immunogenic in mice and rabbits (36).Groups of 5-10 mice receive two subcutaneous injections of VNI-PA-Rcomplex on day 0 and 21 following a protocol analogous to that describedfor vaccination of rats with VNI-PA. Adjuvants are not employed in theseexperiments. Serum samples are collected prior to priming as well asbefore and after boosting. Control animals are injected with VNI alone,vehicle alone, or RTA alone. Serum anti-RTA and anti-PA antibody levelsand their toxin neutralization capacity are determined. One week afterthe boost (day 28) mice are challenged with an i.p. injection of 100ng/g mouse of ricin (Inland Laboratories). This dose corresponds to10-fold the LD₅₀ of ricin. Weights and death of the animals aremonitored daily for 10 days. If animals survive ricin challenge, theexperiment id repeated but mice are challenged 3-4 weeks after a singleinjection. The vaccination dose for a single injection is increased atleast twofold.

The antibody response to vaccination with VNI-PA-R is measured byindirect ELISA. Specifically, serum samples collected prior, during andafter vaccination are tested for the presence of antibodies against RTA,PA, ANTXR2, and FHV, the protein components of the VNI-PA-R complex.ELISA to measure anti-PA, anti-ANTXR2 and anti-FHV are done as describedpreviously. To measure the anti-RTA response, 96-well plates are coatedovernight with RTA (Vector Laboratories), blocked and incubated withserial dilutions of antiserum. Bound antibody is visualized usingcommercially available anti-mouse secondary IgG.

For vaccination with VNI-PA-B complex, groups of 5-10 mice receive twosubcutaneous injections of VNI-PA-B complex on day 0 and 21 following aprotocol analogous to that described for vaccination of rats withVNI-PA. Adjuvants are not employed in these experiments. Serum samplesare collected prior to priming, as well as before and after boosting.Control animals are injected with VNI alone, BoNT(A) heavy chain alone(Metabiologics) or vehicle. Anti-BoNT(A) antibody levels and their toxinneutralization capacity is determined and one week after the secondinjection mice are challenged with an i.p. injection of 5-10 LD₅₀ BoNT/A(Metabiologics). Animals are monitored for signs of botulism overnightand the time to death/survival is recorded. If animals survive BoNT/Achallenge, the experiment is repeated but mice are challenged 3-4 weeksafter a single injection. The vaccination dose for a single injection isincreased at least twofold.

The antibody response to vaccination with VNI-PA-B is measured by ELISA.Specifically, serum samples collected prior, during and aftervaccination will be tested for the presence of antibodies to BoNT(A),PA, ANTXR2 I-domain, and FHV, the protein components constituting theVNI-PA-B complex. ELISA to measure anti-PA, -ANTXR2 I-domain, and -FHVis done as described previously. To measure anti-BoNT/A response,96-well plates will be coated overnight with BoNT/A heavy chain(Metabiologics), blocked and probed with serial dilutions of antiserum.Bound antibody is visualized using commercially available anti-mousesecondary IgG.

Test antibodies raised against VNI particles coated with PA-R or PA-B toneutralize anthrax lethal toxin, ricin or BoNT(A). The IgG antibodiesraised in mice immunized subcutaneously with VNI particles coated withthe PA fusion protein(s) are screened by cell culture-based toxinneutralization assays (TNAs) for their abilities to neutralize anthraxlethal toxin, ricin, or BoNT(A). The anthrax lethal toxin TNA isdescribed above. The ricin TNA involves incubating serial dilutions ofantiserum and ricin holotoxin (Inland Laboratories) prior to theiraddition to Vero cells. Cell survival is subsequently monitored usingthe MTT proliferation assay kit from the ATCC (37).

The BoNT(A) TNA involves mixing serial dilutions of antiserum andBoNT(A) (Metabiologics) prior to their addition to Neuro-2A cells.BoNT(A) activity is then monitored by subjecting protein lysates fromthe cells to SDS-PAGE and then immunoblotting with anti-SNAP25 antibody(Santa Cruz Biotechnology) and HRP-conjugated secondary antibody todetect the toxin cleaved form of SNAP-25 (38). Taken together thesestudies can be used to determine whether VNI particles coated withrecombinant PA-R and PA-B fusion proteins can invoke the production ofneutralizing antibodies against more than one toxin component. Moreover,by immunizing mice with a cocktail of VNI-PA, VNI-PA-R, and VNI-PA-Bparticles, or with VNI particles coated with different ratios of all 3forms of the PA protein, it should be possible to generally vaccinateagainst these biowarfare agents.

Determining which steps in intoxication are blocked by neutralizing IgGantibodies raised against VNI-PA-R and VNI-PA-B. Assuming that the VNIparticles coated with PA-R or PA-B give rise to neutralizing antibodiesagainst more than one toxin component it is useful to identify thefunctional classes of these antibodies. Therefore, they will becharacterized to define the step of anthrax lethal toxin, ricin, andBoNT(A) intoxication which is the target for the neutralizing responses.The studies involving anthrax lethal toxin will be conducted asdescribed above. The studies involving BoNT(A) employ the previouslydescribed immunofluorescence microscopy approach involving fixedNeuro-2A cells to discriminate between those antibodies which blocktoxin binding to cells from those that block toxin uptake (38).Similarly, immunofluorescence microscopy is used to monitor the effectsof neutralizing antibodies on ricin uptake into cells via endocytosisfollowed by retrograde transport to the Golgi apparatus and endoplasmicreticulum prior to translocation to the cytosol, as described previously(39). Antibodies specific for ricin, for the endosomal marker EEA1, forthe Golgi marker TGN46, and for the endoplasmic reticulum marker BiP areemployed for these studies.

Performing in vivo spore or toxin challenge after immunization withVNI-PA vs. VNI-PA-R or VNI-PA-B. Once induction of immunity has beenestablished, A/J mice will be challenged with B. anthracis Sternespores, or with ricin toxin or botulinum toxin. Animals are inoculatedi.p. with 4×10⁵ spores and the lethality determined between 2-14 dayspost-inoculation. A duplicate set of animals are tested to determinewhether protection from either ricin or BoNT(A) challenge occurs, asdescribed above.

Determining whether combination or cocktail of particles can induceneutralizing immunity to all three pathogens. This experiment determineswhether the simultaneous induction of immunity to all three pathogensoccurs. To achieve this, two approaches can be used. First, a “cocktail”of particles, mixing and equal quantity of VNI-PA-R and VNI-PA-B areused to immunize a large group of animals and, subsequently, subgroupsare challenged with each of the three pathogens. In a second approach, a“chimeric” particle is generated where a mixture PA-R and PA-B arearrayed onto VNIs, to generate particles that display all three antigenstogether. A similar immunization and challenge strategy is thenperformed.

Example 2 References

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While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1. An immunogenic composition comprising a nodavirus-derived virus likeparticle (VLP) that comprises a heterologous immunogen binding domain,which binding domain is bound to a first heterologous immunogen, whereinthe binding domain comprises a VWA domain of capillary morphogenesisprotein 2 (ANTRX2) and the heterologous immunogen is derived from ananthrax toxin.
 2. The immunogenic composition of claim 1, wherein theheterologous immunogen comprises anthrax protective antigen (PA).
 3. Theimmunogenic composition of claim 1, wherein the VLP comprises 180binding domains coupled to a maximum of 120 heterologous immunogens. 4.The immunogenic composition of claim 1, wherein the heterologousimmunogen comprises a first domain and a second domain, wherein thefirst domain is heterologous to the second domain.
 5. The immnogeniccomposition of claim 4, wherein the VLP comprises a plurality ofheterologous immunogen binding domains on the surface of the VLP,wherein a second heterologous immunogen is bound to one of theheterologous immunogen binding domains, wherein the second heterologousimmunogen is different than the first heterologous immunogen.
 6. Theimmunogenic composition of claim 5, wherein the second heterologousimmunogen comprises a first and a second domain, wherein the firstdomain is heterologous to the second domain, and wherein the seconddomain of the second heterologous immunogen is different than the seconddomain of the first heterologous immunogen.
 7. The immunogeniccomposition of claim 6, wherein the first domain of the first and secondheterologous immunogen comprises a domain derived from anthraxprotective antigen, and the second domain of the first heterologousimmunogen comprises a domain derived from botulinum A toxin and thesecond domain of the second heterologous immunogen comprises a domainderived from ricin toxin.
 8. The immunogenic composition of claim 1,wherein the VLP is derived from Flock House Virus (FHV).